urinary trypsin inhibitor reduces the release of histamine from rat peritoneal mast cells

Urinary trypsin inhibitor reduces the release of histamine from rat peritoneal mast cells

HIROSHI KOBAYASHI, KIYOSHI SHIBATA, MICHIO FUJIE, and TOSHIHIKO TERAO

HAMAMATSU, JAPAN

We determined the ability of urinary trypsin inhibitor (UTI), which is a Kunitz-type protease inhibitor present in serum and in urine, to inhibit rat peritoneal mast cell (RPMC) mediator release induced by several stimuli. UTI at tenuated the immunoglobulin E-mediated release of both preformed (histamine) and newly formed (leukotriene C4) mediators from RPMCs. Inhibition (21% + 5%) of the anti- IgE-triggered release of histamine was observed after a 30-minute incubation of RPMCs with UTI (5 gmol/L). To investigate the specificity of the UTI effect, we studied the stimulatory activity of phorbol ester (phorbo112-myristate 13-acetate [PMA]) or calcium ionophore A23187 in control and UTl-treated mast cells. The eff icacy of UTI as an inhibitor was dependent on the nature of the stimulus, because histamine release induced by PMA-mediated or calc ium ionophore A23187-mediated processes was not inhibited by UTI. A series of structurally distinct protease inhibitors did not inhibit IgE-induced release of mediators from RPMCs. The Kunitz-type protease inhibitors are important in the regulation of RPMC function. In parallel with the UTl-related decrease in anti-lgE stimulatory activity on mediator release, increased microviscosity of membrane lipids could be observed by two independent experiments on fluorescence polarization with diphenylhexatriene (DPH) and on the fluorescence probe fluorescein isothiocyanate-concanavalin A. UTI reduces mediator release by a mechanism--possibly an interruption of the coupl ing of receptor and effector systems--because UTI acts as an agent to decrease biologic lipid membrane fluidity. (J Lab Clin Med 1998; 131:375-84)

Abbreviations: BSA = bovine serum albumin; Con A = concanavalin A; DPH = diphenylhexatriene; FITC = fluorescein isothiocyanate; HEPES = N-2-hydroxyethylpiperazine-N-2-ethanesul- fonic acid; IgE = immunog[obulin E; IP 3 = inositol 1,4,5-trisphosphate; IT1 : inter-cdrypsin inhibitor; LTC 4 = leukotriene C4; PAI-I = plasminogen activator inhibitor type I; PKC = protein kinase C; PMA = phorboJ 12-myristate 13-acetate; RIA = radioimmunoassay; RPMC = rat peritoneal mast cell; SBTI = soybean trypsin inhibitor; UTI = urinary trypsin inhibitor

t has been reported that UTI, which is a Kunitz- type protease inhibitor present in serum and in urine, has an antinvasive and antimetastatic activ-

ity by inhibiting tumor cell-associated plasmin activity. 1-9 UTI inhibits various serine proteases such as trypsin, c~-chymotrypsin, granulocyte elastase, plasmin,

From the Department of Obstetrics and Gynecology, Equipment Cen- ter, Hamarnatsu University School of Medicine. Submitted for publication June 10, 1997; revision submitted Oct. 6, 1997; accepted Nov. 10, 1997. Reprint requests: Hiroshi Kobayashi, MD, Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Han- dacho 3600, Hamamatsu, Shizuoka, 431-31, Japan. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5/1/88221

cathepsin G, and hyaluronidase. 10-16 UTI is linked to the C-terminal of c~ 1-microglobulin via two basic amino acids within the same reading frame. UTI secretes into plasma as the light chain of ITI, which consists of two heavy chains (HC1 and HC2) and one light chain (UTI). 17 In recent years a number of studies have emerged that have investigated the effects of UTI on the function of inhibition of tumor cell invasion and metastasis. 1-9 UTI is thought to act on inhibition of a certain pathway in signal transduction to trigger processes involved in invasion and metastasis.~SJ 9 In addition, UTI inhibits thrombin-induced calcium influx in platelet and endothelial cells.20, 21 The mechanism by which UTI inhibits cell activation is unclear, and sug- gested mechanisms include the inhibition of PKC activity and the modulation of calcium influx.2°, 2l

375

J Lab Clin Med 376 Kobayashi et al. April 1998

It has been reported by several clinical and experimental studies that UTI has protective effects against tubu- lar injury in acute renal failure as well as in patients treated with high-dose cisplatin, suggesting that it is possible that UTI directly stabilizes the lysosomal or plasma membrane of cells. These results suggest that UTI may stabilize membrane lipid bilayers. 22

We speculate that UTI may act as a an agent to decrease the fluidity of biologic lipid membranes. Cell activation is often accompanied by changes in lipid mobility in the plasma membrane. The activation of cells is also accompanied by a change in membrane fluidity. 23-25 Evidence is emerging that structural features of the membrane lipid bilayer may play a role in mod- ulating the activity of PKC, 26 although the exact nature of this property remains to be elucidated.

Mast cells provide a suitable model for studying the mechanisms of cellular activation and exocytosis. 27 These cells can be activated by physical, chemical, and immunologic stimuli. Mast cell activation leads to the release of histamine and other mediators with proinf lammatory and spasmogenic properties. 28-3° It has been reported that the ability of anti-IgE to elicit specific and noncytotoxic histamine release f rom RPMCs. Histamine release from mast cells is initiated by the fusion of the cell membrane and intracellular granule membrane through the mechanism of exocytosis.

In this article we characterize the inhibitory mechanism of UTI that reduces the act ivat ion o f R P M C response to anti-IgE. We show that UTI part ial ly attenuates the anti-IgE-mediated release of both preformed (histamine) and newly formed (LTC4) mediators f rom RPMCs. However , UTI is less effective against mast cells s t imulated with phorbol ester (PMA) and Ca 2÷ ionophore A23187; this is probably related to the differing mechanisms of mast cell activation. One possible site of action of UTI may be the interruption o f receptor-ef fec tor signals. Further- more, the increased microviscos i ty o f membrane lipids could be observed by exposure on UTI. Our results suggest that UTI acts general ly as a potent stabilizer on the biologic lipid bilayers and that it reduces membrane permeabil i ty, relat ing to some changes in the phys icochemica l nature o f biologic membranes.

METHODS

Materials. Aprotinin, soybean trypsin inhibitor, leupeptin, and E64 were purchased from Sigma Chemical Co., St. Louis, Mo. (Zl-Antitrypsin, antithrombin III, and ~2-macroglobulin were obtained from Cosmo Bit Co., Tokyo, Japan. Activated recombinant human PAI-1 was obtained from Wako Pure Chemicals Co., Tokyo, Japan.

Preparation of RPMCs. RPMCs were prepared from the peritoneal fluid of female Wistar rats (180 to 200 gin). 26 Tyrode-HEPES solution (NaC1, 134 mmol/L; NaHCO 3, 12 retool/L; KC1, 2.9 mmol/L; MgC12, 1 retool/L; CaCle, 1.8 mmol/L; NaH2PO 4, 0.36 mmol/L; glucose, 5.6 mmol/L; HEPES, 10 retool/L; 0.1% BSA [wt/vol], pH 7.4, 10 ml) containing 1 U/m1 heparin was injected intraperitoneally, and the abdomen was massaged for 3 minutes. A laparotomy was performed, and the fluid containing cells was removed. The cells were centrifuged at 120 g for 7 minutes. The cells were resuspended in 10 ml Tyrode-HEPES solution. Mast ceils stained with neutral red were counted by using a hemocy- tometer, and samples containing 5 x 105 mast cells in 1 ml were prepared.

U937 promyeloid cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. Ceils were har- vested by centrifugation and used for the FITC-Con A experiments. Growth conditions and maintenance have been previously described. 31

Human monocytes were prepared and incubated as described.32,33

Mediator release experiments. RPMCs (1 x 106 cells) were suspended in 2 ml of Tyrode-HEPES solution and pre- warmed at 37 ° C for 5 minutes. 33,34 Materials to be tested were added to give the total volume of 2.5 ml, and further incubation was performed. When tested for the effect of UTI, the cells were preincubated at 37 ° C for the indicated periods of time with various concetrations of UTI. In experiments that used IgE-mediated activation, the cells were sensitized with 0.1 gg/ml rat IgE for 3 hours at 4 ° C before being challenged with anti-IgE. The cells were then washed three times with Tyrode-HEPES solution and incubated for 30 minutes at 37 ° C with 0.5 ml of anti-IgE (1:300) in the presence of various concentrations of UTI (0 to 10 gmol/L). RPMCs were incubated with or without UTI for time periods as indicated in the text before activation.

In a parallel experiment, the cells were incubated for 30 minutes at 37 ° C with 0.5 ml of either buffer, calcium ionophore A23187 (0.3 gg/ml), or PMA (100 nmol/L). Reac- tions were stopped by the addition of 2.5 ml ice-cold Tyrode- HEPES solution. Supernatants were centrifuged for 3 minutes at 400 g. All experiments were performed in duplicate.

In experiments designed to determine whether other protease inhibitors inhibit mediator release from RPMCs, RPMCs were incubated with protease inhibitors (aprotinin, SBTI, leupeptin, E64, cq-antitrypsin, anti-thrombin III, c~ 2- macroglobulin, and PAI-1) for time periods as indicated in the text before activation. An aliquot of the supernatant was removed and stored frozen for histamine and LTC 4 analysis.

Measurement of histamine and arachidonic acid metabolite release. Liberated histamine (supernatant) was measured by RIA with commercially available assay reagents according to the manufacturer's instructions (Histamine RIA kit; Immunotech, Marseille, France), and concentrations were

J Lab Clin Med Volume 131, Number 4

calculated from a standard curve. Total histamine in cell sus-

pensions was quantified after cell lysis in distilled water and

freeze-thawing. RPMCs incubated in buffer alone served as

a measure of spontaneous histamine release, which ranged

from 2% to 8% of the total histamine content. Incubation of

cells in the absence or presence of UTI

(5 gmol/L) did not affect the spontaneous histamine release

(control, 4% + 2%; plus UTI, 5% -+ 1%; n = 3). Histamine

release was thus expressed as a percent of the total histamine

content after subtracting the spontaneous histamine release.

All experiments were performed in duplicate. At the concen-

trations used, UTI did not interfere within the assay. The limit

of detection for the histamine assay is 0.2 nmol/L. The

intraassay and interassay coefficients of variation for this

method are <8% and <11%, respectively. Histamine levels

were calculated as nmol/104 cells.

LTC 4 was measured by RIA as previously described, 36 with

dextran-coated charcoal as the separation technique (LTC 4-

specific tritium assay system; Amersham Life Science, Tokyo,

Japan). The antibody to LTC 4 cross-reacts 5.4% with LTD 4

and 0.5% with LTE 4. The limit of detection of the LTC 4 RIA

in these experiments was equivalent to 0.1 ng LTC 4 per 106

ceils.

Determination of 1,4,5-1P 3 content. Cells (5 x 105 cells)

cultured were incubated at 37 ° C for 5 minutes in 1 ml of

Tyrode-HEPES solution in the presence or absence of UTI (5

gmol/L) and then stimulated with IgE/anti-IgE (1/1000 dilu-

tion). The reactions were terminated after immediate centrifu-

gation by the addition of 1 ml of ice-cold 2% (wt/vol) per-

chloric acid (HC104). The tubes were placed for 30 minutes

in an ice bath. After centrifugation, the supernatant was sep-

arated and its pH was adjusted to about 7.0 with 1.5 mol/L

KOH solution containing 75 mmol/L HEPES. KC10 4 was

precipitated for 30 minutes at 4 ° C and sedimented by cen-

trifugation. The amount of IP 3 in the final supernatant was

determined by the competitive binding assay (D-myoinositol

1,4,5-trisphosphate tritium assay system; Amersham Life Sci- ence).37-39

Isolation of human protease inhibitors. Human ITI was

isolated as described previously. 4° UTI was isolated from

human urine as described previously. 1-9

Fluorescence polarization measurement. Control or

UTI-treated mast cells were washed, resuspended in phos-

phate-buffered saline solution, incubated for 30 minutes at

37 ° C with 0.6 gmol/L DPH, and then subjected to polariza-

tion analysis with a spectrofluorometer. DPH was excited at

357 nm, and emission was recorded at 430 nm. Steady-state

fluorescence polarization was computed according to the rela-

tionship P = (Ivv-lvh)/(lvv + Ivh), where I~v and lvh are the fluorescence intensities observed with the analyzing polarizer

parallel and perpendicular to the polarizer excitation beam,

respectively. The data were connected for unequal transmis-

sion of differently polarized light and for intrinsic fluorescence.41-43

Kobayashi et al. 377

2s"

20"

'~ ~lS-

"~ ~ lO- ~ ~ . s"

0 -

i

UTI concentration, u M Fig. 1. Dose-dependent effect of UTI on anti-IgE-stimulated histamine release. Mast cells were preincubated with increasing concentrations of UTI (0 to l0 gmol/L) for 30 minutes at 37 ° C, then challenged with anti-IgE (1:300) for a further 30 minutes for histamine release. Results are expressed as the percent inhibition by UTI of control histamine release, which was 25.0% _+ 3.6%. Statistically significant (p < 0.05) levels of inhibition are indicated with an asterisk. Values are expressed as mean _+ SD for three experiments.

Measurement of cap formation. The effect of UTI on

Con A-mediated cap formation in mast cells and promyeloid

leukemia U937 cells was examined. The cells were preincu-

bated with various concentrations of UTI for 30 minutes at

37 ° C and further incubated for 30 minutes by adding 10

gg/ml FITC-Con A, were washed twice in a cold phosphate-

buffered saline solution containing 0.1% BSA, and were

examined for surface staining with a fluorescence microscope

and for fluorescence intensity by flow cytometry. 43

Statistical evaluation of data. Differences in mediator

release were assessed by paired Student's t test. Results were

considered significant at p < 0.05.

RESULTS

Effect of UTI on mediator release from RPMCs. We deter-

mined the ability of UTI to inhibit RPMC mediator

release induced by anti-IgE. Unst imulated mast cells

produced negligible amounts of histamine (4% + 2%)

when incubated for periods up to 1 hour. However, mast

cells stimulated with anti-IgE and incubated for 30 min-

utes caused a significant release of histamine. Anti-IgE

acted in a concentrat ion-dependent manner (data not

shown; see references 44 and 45).

The effect of UTI on anti-IgE st imulation of hista-

mine release in RPMCs is shown in Fig. 1. Mast cells

were challenged with IgE followed by anti-IgE after 30

minutes of exposure to Tyrode-HEPES buffer alone or

buffer containing various concentrations of UTI (0 to

10 gmol/L). Histamine release was measured by RIA.

There was a significant decrease in anti-IgE-stimulated

histamine release in RPMCs preincubated for 30 min-

utes at 37 ° C with increasing UTI concentrations up to

10 gmol /L (Fig. 1). UTI reduced the release of hista-

mine in a dose-dependent manner with significant (p <

0.05) levels of inhibit ion at concentrat ions of >5

378 Kobayashi et al.

2S"

20"

"6~i s. ~=o~0.

~ s

8 i"o 2'0 ~o 4'o s'o 6b Preincubation time, min

Fig. 2. Time course of UTI-induced inhibition of anti-IgE-stimulated histamine release. Mast cells were preincubated for the indicated time (0 to 60 minutes) with 5 gmollL UTI. Then the cells were incubated with anfi-IgE (1:300) at 37 ° C for 30 minutes in the presence of UTI, and histamine release was measured by RIA. Results are expressed as the percent inhibition by UTI of control histamine release, which was 24.3% _+ 4.1%. Statistically significant (*p < 0.05) levels of inchibition were obtained after incubation times of 20 minutes or longer. Values are expressed as mean + SD for three experiments.

gmol/L. It is possible to conclude from the above results that the release of histamine was reduced by UTI rather than to conclude that released mast cell histamine was neutralized or degraded. However, the basal levels of the histamine release remained unchanged by UTI treatment.

The time course of UTI-induced inhibition of anti- IgE-mediated histamine release was examined (Fig. 2). Mast cells were preincubated with 5 gmol/L UTI for between 5 and 60 minutes before challenge with anti- IgE. UTI-induced inhibition of histamine release was first detectable after 5 minutes, and inhibition reached 23 % + 3 % after 30 minutes of preincubation. Prolonged exposure to UTI is not required before an inhibitory effect on anti-IgE-mediated histamine release can be appreciated.

In the preceeding experiments, RPMCs were preincubated with UTI, and the UTI was present during the challenge step with anti-IgE. Further studies have indicated that for the UTI to attenuate mediator release, it was necessary to maintain RPMCs with UTI during the challenge step (Fig. 3).

To investigate the specificity of the UTI effect with regard to other stimulatory systems, we studied the stimulatory activity of PMA (100 nmol/L) or calcium ionophore A23187 (0.3 gg/ml) in control and UTI-treated mast cells. We used several compounds to directly activate two of the main intracellular mechanisms responsible for mast cell degranulation (the activation of PKC and the increase in intracellular Ca 2+ concentration), thus bypassing some of the early stages of the receptor-mediated release process; the Ca 2+ ionophore A23187 increases the intracellular Ca 2+ concentra-

J Lab Clin Med April 1998

25

20

0 0

i s .'N_o ~._~

e-

0

unwashed washed Fig. 3. Effect of washing on the inhibition of histamine release by UTI. Ceils were preincubated with UTI (5 gmol/L) for 30 minutes at 37 ° C and then either left untreated (unwashed) or were washed with buffer to remove extracellular UTI (washed). The ceils were then challenged with anti-IgE (1:300) for a further 30 minutes for the release of histamine. Values are expressed as mean + SD for three experiments. *p < 0.05.

tion, 46 whereas phorbol ester such as PMA activates isoforms of PKC. 47

The effect of UTI on net histamine release by secretagogues (anti-IgE, PMA, and A23187) is shown in Table I. Phorbol ester alone released less than 20% of total cellular histamine, and the calcium ionophore A23187 markedly stimulated histamine release by directly elevating free intracellular calcium levels. The histamine release elicited by anti-IgE with 5 gmol/L UTI is significantly inhibited when compared with results with anti-IgE alone (p < 0.05). Cell tre~/tment with 5 gmol/L UTI, however, did not modify PMA activity on histamine release. PMA directly stimulates PKC activity, which has a regulatory domain that inserts into the membrane hydrophobic region. Also, UTI showed no protective effects on the ionophore.

A number of structurally distinct protease inhibitors (aprotinin, SBTI, cq-antithrombin, c~2-macroglobulin, antithrombin III, PAI-1, leupeptin, and E64) were also investigated for inhibitory effects on histamine release induced by anti-IgE, PMA, and the ionophore A23187 (Fig. 4). UTI has the Kunitz-type protease inhibitor domain, which may be important for inhibition of the stimulated release of mediators from RPMCs. We therefore tested the effects of Kunitz-type protease inhibitor members on its postulated role in mediator release. RPMCs were incubated for 30 minutes at 37 ° C with each protease inhibitor (5 gmol/L) and then challenged with anti-IgE (1:300), PMA (100 nmol/L), or ionophore

J Lab Clin Med Volume 131, Number 4

Table I. Effect of uTI on histamine release from mast cells induced by anti-lgE, PMA, and A23187

Secretagogues Histamine release (concentration) (percent total cellular histamine)

-UTI +UTI

Buffer 4.0 + 0.1 5.3 _+ 0.8 Anti-lgE (1:300 dilution) 32.3 • 2,9" 24.2 -+ 2.2* PMA (100 pmol/L) 15.7 ~- 1.8 15.2 _+ 2,2 A23187 (300 ng/ml) 39.1 + 1.7 41.1 _+ 1.3

Purified mast cells were washed, equilibrated, sedimented, and then resuspended in Tyrode-HEPES solution. Cells were aliquoted and were stimulated by the addit ion of the indicated releasing agents, Mast cells were chal lenged with anti-lgE (1:300 dilution) and with non-lgE-mediated secretagogues (PMA [100 nmol/L] and A23187 [0.3 i~g/ml)) to probe the effects of UTI on different biologic pathways of activation of these cells. Cells were incubated for 30 minutes at 37 ° C with UTI (5 pmol/L) and then challenged (30 minutes, 37 ° C) with PMA or calcium ionophore A23187 in the presence of 5 gmol/L UTI, The values represent the mean percentages of triplicates + SD of total mast ceil histamine. Simi- lar results were obtained in experiments of similar design on two other occasions,

*p < 0.05,

A23187 (0.3 gg/ml). Of the protease inhibitors, UTI, aprotinin, and SBTI were effective at inhibiting the stimulated release of mediators from RPMCs, whereas members of the non-Kunitz-type protease inhibitor family were virtually ineffective. Again, histamine release induced by either PMA or A23187 was unaf- fected by pretreatment with any protease inhibitors. These results suggest that non-Kunitz-type inhibitors were found to be ineffective as inhibitors of histamine release.

To determine whether the effects of UTI were restricted to the modulation of the preformed mediator histamine, the effect of UTI on the IgE-mediated generation of LTC 4 (newly formed mediator) was investigated. UTI was also found to reduce the generation of LTC 4 in a dose-dependent manner (Fig. 5).

Anti-IgE-mediated activation of mast cells is closely coupled with phospholipid metabolism. We investigated whether UTI has an inhibitory effect on membrane phospholipid turnover stimulated by anti-IgE (Fig. 6). Anti-IgE-induced rapid and transient generation of IP 3 was significantly suppressed by UTI. Preincubation with 5 gmol/L UTI resulted in a dose-related inhibition of anti-IgE-mediated (1/300 dilution) IP 3 generation that was maximal at >5 gmol/L. UTI is ineffective at concentrations less than 5 gmol/L (data not shown). As shown in Fig. 6, anti-IgE-induced IP 3 accumulation was reduced to approximately two thirds by 5 gmol/L of UTI. UTI significantly suppressed the formation of inositol 1,4,5-trisphosphate.

Kobayashi et al. 379

To characterize the effect of anti-IgE on mast cell IP 3 formation, concentration response curves were assessed for anti-IgE (Fig. 6, inset). In addition, we examined whether UTI is more effective at lower stimulation levels. At all concentrations of anti-IgE tested, the inclusion of UTI (5 gmol/L) decreased anti-IgE- mediated IP 3 at 1 minute. UTI may change the medi- an effective concentration of anti-IgE for IP 3 formation, which is 1/3000 dilution or 1/10,000 dilution of anti-IgE in the presence or absence of UTI, respectively. It is likely that UTI is more effective at lower stimulation levels of antMgE. In a separate experiment, we confirmed that UTI inhibited the mobilization of cytosolic calcium ions (data not shown; see references 20 and 21).

Effect of UTI on plasma membrane fluidity. It has been recognized that the histamine release from mast cells is initiated by the fusion of cell membrane and intracellular granule membrane--exocytos is . 48 Previous evidence exists to suggest that membrane fluidity affects histamine release by mast cells. 23,42,43,48 The alteration in membrane fluidity was estimated to investigate whether UTI should alter the physicochemical nature of biologic membranes, although it is unlikely that UTI may rapidly penetrate the phospholipid bilayers of cellular membranes because of its high molecular weight. We investigated the regulatory mechanism of the fluidity of cell surface membranes by UTI. To assess whether UTI can alter the physicochemical nature of biologic membranes, we used two independent methods to estimate membrane fluidity: (1) measurement of the degree of polarization of DPH in the cell membrane; (2) microscopic observation of patch and cap formation of FITC-Con A on the cell surface.

In parallel with the UTI-related decrease in anti-IgE stimulatory activity on histamine release, an increased microviscosity of membrane lipids could be observed through experiments on fluorescence polarization with DPH (Fig. 7). The addition of UTI to the cells may decrease lipid fluidity of the outer membrane leaflet, thus showing a stiffening action that can alter many of the dynamic membrane features by modification of specific intermolecular interactions. UTI may modify many biologic actions on the cell membrane by sup- pressing membrane fluidity.

When RPMCs, human peripheral blood monocytes, or U937 promyeloid leukemia cells (Table II and Fig. 8) were incubated with FITC-Con A at 37 ° C, patch formation was observed in the cell surface at the initial phase of the incubation, and then these patches changed to the cap formation (Fig. 8). When these cells were preincubated with various concentrations of UTI for 30 minutes at 37 ° C, cap formation was markedly sup-

J Lab Clin Med 380 Kobayashi et al, April 1998

2 S. ~ anti-lgE

I l l P.A a0. ~-

~) 1AZ3187 I D

c ' ~ 15. O t...

~_-.__

~1o-~= s

0 "1-- I I

UYl apro. SBTI ' a lAT ' a2M ATIII PAl-1 keup. E64 I i i f

Kunitz-type Non-Kunitz-type

Fig. 4. Effect of Kunitz-type and non-Kunitz-type protease inhibitors on histamine release from RPMCs. Cells were preincubated with each protease inhibitor (5 gmol/L) for 30 minutes at 37 ° C and then challenged with either anti-IgE (1:300), PMA (100 nmol/L), or A23187 (0.3 gg/ml) for a further 30 minutes at 37 ° C for histamine release. Results are expressed as the percent inhibition by the compounds of control histamine release, which were 23.1% + 6.3% for anti-IgE, 31.2% + 5.3% for PMA, and 25.3% _+ 4.9% for A23187. Values are expressed as the mean from two experiments. The results of a single experiment, representative of two, are shown.

2S'

20'

~Ols.

C ~ S

~H0 -5

UTI concentration, /~ M § 9 I0

Fig. 5. Effect of UTI on the generation of LTC 4 from stimulated RPMCs. RPMCs were preincubated for 30 minutes at 37 ° C with increasing concentrations of UTI (0 to 10 gmol/L) and then challenged with anti-IgE (1:300) for a further 30 minutes for LTC 4 generation. Results were expressed as the percent inhibition by UTI of LTC 4 generation. Control LTC 4 generation was 12 _+ 4 rig/106 cells. Statistically significant (p < 0.05) inhibition is indicated with an asterisk. Values are expressed as mean _+ SD for three experiments.

pressed in a dose-dependent manner. These results strongly suggest that UTI may act generally as a potent stabilizer on the biologic lipid bilayers and may reduce membrane fluidity.

We speculated that membrane fluidity is associated with the exocytotic machinery of mast cells. Accord- ing to the results described in Figs. 7 and 8, UTI likely suppressed membrane fluidity. Therefore the reason histamine release increased by PMA or A23187 was not inhibited is that the inhibitory effects of UTI on receptor-mediated responses can be bypassed by use of phorbol ester and calcium ionophore.

DISCUSSION

The activation of mast cells results in the release of inflammatory mediators such as histamine and leukotrienes. 23 Histamine release was achieved by bypassing the cell surface receptors with Ca 2÷ ionophores to elevate the cytosolic Ca 2+ concentrations, by bypassing the receptors with phorbol esters to activate intracellular PKC (after cross-linking of membrane-bound IgE via anti-IgE), or after binding of the chemoattractant formyl-methionyl-leucyl-phenylala- nine. 25-29,34-39 In general, histamine release is intimate- ly connected with the formation of vacuoles, which may

J Lab Clin Med Volume 131, Number 4 Kobayashi et al, 381

1 2 '

10' ~ 8

0 0 ~ 6 ' u m 0 O- E

4 ' ~ n

anti-lgE

ti-lgE + UTI

Time, min

Fig. 6. Effect of UTI on anti-IgE-induced IP 3 formation in mast cells (time-dependent changes in IP 3 concentration). Cells (5 x 105) were preincubated at 37 ° C for 5 minutes in 1 ml of Tyrode-HEPES solution and then stimulated with anti-IgE (1:1000) for the indicated periods of time. Cells were stimulated with buffer alone (O), anti-IgE (0), or anti-IgE plus 5 gmol/L UTI (A). IP 3 content was measured as described under Methods. The peak of anti-IgE-induced IP 3 at 1 minute was reproducible. UTI (5 gmol/L) suppressed anti-IgE- induced production of IP 3 at 1 minute after anti-IgE addition. Inset, The peak value of IP 3 at 1 minute after the addition of various concentrations of anti-IgE was decreased by 5 gmol/L of UTI. Bar indi- cates SD. The mean _+ SD of three experiments is shown. *p < 0.05.

be the necessary prerequis i te for the occurrence of structural changes in granules.

Differences in the cell activation pathways tr iggered by these st imuli were ana lyzed with UTI. His tamine release induced by ant i - IgE was inhibi ted by 20% to 25% by UTI (5 gmol /L) . Ant i - IgE, which causes arachidonic acid metabol i sm, induced the generat ion of LTC 4, and the generation of IP 3 was also reduced by 5 gmol /L UTI. Inhibition required at least 5 minutes of UTI exposure. However, UTI is ineffective against mast cells st imulated with P M A or A23187; this is probably related to the differing mechanisms of mast cell activation. The lack of effect of UTI on PMA- and A23187- mediated release suggests that UTI may act at the level of the receptor /ef fec tor system, because P M A and A23187 bypassed receptor-mediated events. Inasmuch as antigen stimulates phospholipase A 2 and phospholipase C through G proteins, the data s t rongly suggest that UTI may interrupt the coupling between receptors and effector systems, perhaps through effects on G proteins .49,50

UTI may suppress the 1 ,2 -d iacy lg lycero l accumulat ion, cons ider ing the resul ts of IP 3 fo rmat ion and Ca 2+ mobil izat ion. Ca a+ plays a crucial role in the reg- u la t ion of the secre tory response or s ignal t ransduc- tion in many types of cells including mast cells. 46 As previously observed in human umbil ical vein endothe- l ial cells, UTI suppressed the accumulat ion of [Ca2+] i in t h rombin - s t imu la t ed cells . The [Ca2+]i r e sponse consis ted of two components , (1) the ini t ial t ransient

S -

Q. ~ 3

• ~ z o .u

experiment 1

UTI concentration, ~ I~ Fig. 7. Dose-dependent effect of UTI on microviscosity of membrane lipids. Mast cells were preincubated with various concentrations of UTI (0 to 10 gmol/L) for 30 minutes at 37 ° C, then assayed for microviscosity (©). Another experiment gave a similar result (0). The change in microviscosity of membrane lipids was determined by experiments on fluorescence polarization with DPH. This probe is widely used in studies of membrane fluidity because of its hydrophobic nature and its location in the center of the lipid bilayer with its symmetry axis normally to the plane of the membrane. The results of two experiments are shown.

increase independent of extracellular Ca 2+, and (2) the subsequent susta ined increase caused by influx f rom the external medium. UTI dec reased both in ternal release and influx. 20,21 The results obtained in the present s tudy c lear ly indica te that UTI suppresses anti- IgE- induced IP 3 formation at the same concentrat ions that inhibit the release of histamine. The inhibit ion of both IP 3 formation and the initial transient increase in [Ca2+] i by UTI indica tes that UTI may prevent the activation of PLC. These f indings permit one to con- template mult iple inhibitory effects on each of the sig- na l - t r ansduc ing enzymes . I t is most l ike ly that UTI b locks the s ignal - t ransducing pa thway be tween antigen binding to the cells and the G protein/PLC activation.

The binding of ant i - IgE to the mast cell membrane may produce alteration of membrane lipoprotein structure, leading to changes in membrane permeabili ty/flu- idi ty and enabl ing the entry of ext racel lu lar ions and water, which induce cel lular swell ing. 23,42,43 The

inhibi tory effect of UTI was accompan ied by an increase in membrane microviscosi ty, as est imated by measurements of f luorescence polar izat ion. 42 These data suggest that UTI diminishes the coupling efficacy be tween ant i - IgE receptor complex and effectors because of an increase in p lasma membrane rigidity. Thus these data suggest that UTI has two possible sites of action: (1) the inhibi t ion of media tor re lease by a mechanism, poss ib ly the interrupt ion of the coupl ing of receptor and effector systems; (2) the action of UTI as a decreasing agent of b io logic l ipid membrane flu-

idity. It is not yet known whether any target enzymes of

382 Kobayash i e t al. J Lab Clin M e d

Apri l 1998

Fig. 8. Effect of UTI on the cap formation of FITC-Con A on U937 ceils. U 937 cells were preincubated with various concentrations of UTI (5 gmol/L [A]; 2 gmol/L [B]; 1 gmol/L [C]; and 0 gmol/L [D]) for 30 minutes at 37 ° C, and FITC-Con A (10 gg/ml) was added and incubated for an additional 30 minutes, and the cells in cap formations were counted by microscopic photographs. Cells formed a fluorescent ring in the presence of UTI.

Table II. Inhibition of cap formation by UTI

Cap formation Flow cytometry UTI (% of total cells) FMC

Cells (gmol/L) (mean _+ SD; n = 4) (mean _+ SD; n = 3)

U937 leukemia cells 0 68 _+ 8 263 __ 18 1 67__ 12 257_+ 11 2 44 _+ 1 O* 173 _+ 23* 5 24 _+ 6* 103 _+ 8*

Human monocytes 0 61 _+ 11 219 _+ 17 1 59_+6 217_+15 2 41 _+ 13" 152 _+ 10" 5 20 _+ 8* 69 _+ 7*

Rat peritoneal mast cells 0 58 + 13 297 _+ 9 1 64 -+ 16 260 _+ 1 3 2 49 + 15" 188 -+ 11" 5 25 -+ 7* 121 _+ 5*

The ef fect of UTI on Con A -med ia ted cap format ion in promyeloid leukemia U937 cells, human monocytes, and RPMCs was examined. The cells were preincubated with various concentrations of UTI (0, 1,2, and 5 timol/L) for 30 minutes at 37 ° C, further incubated for 30 minutes by adding 10 gg/ml FITC-Con A, washed twice, and examined for surface staining with a f luorescence microscope and for fluorescence intensity by flow cytomet~/. *p < 0.05 as compared with the control (UTI = 0 gruel/L).

UTI are present in mast cells and whether the activities of the enzymes are inhibited by UTI. Mast cell degranulation results in the release of serine class proteases with trypsin- and chymotrypsin-like specificity (tryptase and chymase). 51 Both trypsin and chymotrypsin can be inhibited by UTI. However, human

UTI is unable to inhibit human tryptase and human chymase. 52 Furthermore, these proteases are also not inhibited by a fragment that consists only of the trypsin-specific C-terminal inhibitor domain of human UTI (HI- 8). The existence of a tryptase-specific low-molecular- mass protease inhibitor (trypstatin) in RPMCs has only

J Lab Clin Med Volume 131, Number 4 Kobayashi et al, 383

recently been claimed. 52 Trypstatin is considered to be identical to HI-8. 52 In human beings, tryptase and chymase activities themselves are not physiologically reg- ulated by UTI and HI-8.

Tryptase and trypstatin are found in the same granules of mast cells. 51 It has been reported that bovine tryptase and trypstatin form a complex and are local- ized within the mast cell granules, where they occur in clusters; this may be taken as an indication of their interaction in vivo and suggests a physiologic role for bovine trypstatin in the regulation of tryptase prote- olytic activity. 51 However, human tryptase is insensi- tive to human trypstatin. 52 This observation prompted us to investigate to a deeper detail the physiologic role of UTI in mast cell degranulation. This is the first report of the pharmacologic manipulation of the activity of UTI on mediator release. UTI may reduce mast cell-dependent mediator release as a secondary consequence of stabilizing various cell membranes. Endogenous trypstatin may act as a potent membrane- stabilizing agent in an autocrine or paracrine fashion immediately after stimulation and activation of RPMCs. It is likely that, besides being a protease inhibitor, trypstatin released from activated mast cells functions to inhibit (regulate) mediator release from neighboring mast cells. The precise mechanism by which UTI inhibits cell activation is unclear, but sug- gested mechanisms include the interruption of the coupling of receptor and effector systems, followed by the inhibition of PKC and the modulation of calcium influx.

In summary, this is the sequel to our publications on the effects of UTI on various types of cells. In this article we have examined the effects of UTI on mast cells. We carried out two series of experiments, one dealing with the effects of UTI on mast cell functions and the other dealing with the effects of UT! on membrane fluidity. UTI modestly attenuates (approximately 20%), in a concentration-dependent (Fig. 1) and time-dependent (Fig. 2) manner, the FceRI-mediated release of histamine and LTC 4 (Fig. 5) from RPMCs. This attenua- tion is associated with a reduction in IP 3 levels (Fig. 6), decreased receptor capping (Fig. 8 and Table II), and increased viscosity of plasma membranes (Fig. 7). As in other cells, inhibitory effects of UTI on receptor-mediated responses can be bypassed by use of phorbol ester and calcium ionophore. We speculate that UTI acts on events upstream of PKC (as noted in previous papers; see reference 18) and reduces the coupling efficacy of FceRI with effectors, possibly as a consequence of increased membrane viscosity (Figs. 7 and 8; Table II). The major concerns are (1) the significance of the 20% reduction in the responses of stimulated (by anti-IgE) mast cells and (2) the

concentration of UTI (5 gmol/L) needed to achieve this reduction. In previous studies 18 we achieved 60% or greater inhibition of receptor-mediated responses in endothelial cells, platelets, and U937 cells with lower concentrations of UTI than those used here. For example, tumor necrosis factor-induced expression of plasminogen activator in U937 and endothelial cells was inhibited approximately 50% with 0.1 gmol/L UTI and was inhibited nearly maximally with 1 gmol/L UTI. These concentrations were consistent with the specific binding of UTI to U937 cells, with maximal binding of 0.2 to 0.3 gmol/L. As for the specific binding of UTI to these cells, the studies in which mast cells are used to study the mechanism of UTI action may be affect- ed by the previous one, in which tumor necrosis factor-induced expression of plasminogen activator in U937 cells and endothelial cells was inhibited with UTI. Although UTI specifically binds to certain tumor cells and endothelial cells, UTI is unlikely to bind to mast cells (unpublished data; Kobayashi et al, 1997). Although our previous observations in tumor and endothelial cells are interesting and wor- thy of further study, the present observations indi- cate that the mast cell may not be the most appropri- ate model. Without the evidence for target enzymes, it is difficult to interpret the effect of UTI on cellular function. The relevance to the therapeutic effects of UTI is questionable in view of the small effects. More-potent analogs or compounds need to be devel- oped if clinical efficacy is contemplated. We are now attempting to increase this histamine release- inhibitory activity by substituting some amino acids in UTI or its derivatives (truncated forms) by using recombinant DNA technology.

However, it is tempting to speculate on the relevance of these findings to the clinical use of UTI and its derivatives as well as their modification in the treatment of allergic reaction, inflammation, and malignancy. An agent such as UTI that inhibits the release of proteases, histamine, and cytokines from activated cells could therefore be beneficial in the treatment of inflammation and cancer.

REFERENCES

1. Kobayashi H, Gotoh J, Hirashima Y, Terao T. Inter-alpha- trypsin inhibitor bound to tumor cells is cleaved into the heavy chains and the light chain on the cell surface. J Biol Chem 1996;271:11362-7.

2. Kobayashi H, Shinohara H, Fujie M, Gotoh J, Itoh M, Takeuchi K, et al. Inhibition of metastasis of Lewis lung car- cinoma by urinary trypsin inhibitor in experimental and spontaneous metastasis models. Int J Cancer 1995;63:455-62.

3. Kobayashi H, Shinohara H, Gotoh J, Fujie M, Fujishiro S, Terao T. Anti-metastatic therapy by urinary trypsin inhibitor

384 Kobayashi et al,

in combination with an anti-cancer agent. Br J Cancer 1995;72:1131-7.

4. Kobayashi H, Gotoh J, Kanayama N, Hirashima Y, Terao T, Sugino D. Inhibition of tumor cell invasion through matrigel by a peptide derived from the domain II region in urinary trypsin inhibition. Cancer Res 1995;55:1847-52.

5. Kobayashi H, Gotoh J, Hirashima Y, Fujie M, Sugino D, Terao T. Inhibitory effect of a conjugate between human urokinase and urinary trypsin inhibitor on tumor cell invasion in vitro. J Biol Chem 1995;270:8361-6.

6. Kobayashi H, Gotoh J, Fujie M, Terao T. Characterization of the cellular binding site for the urinary trypsin inhibitor. J Biol Chem 1994;269:20642-7.

7. Kobayashi H, Fujie M, Shinohara H, Ohi H, Sugimura M, Terao T. Effects of urinary trypsin inhibitor on the invasion of reconstituted basement membranes by ovarian cancer cells. Int J Cancer 1994;57:378-84.

8. Kobayashi H, Shinohara H, Ohi H, Sugimura M, Terao T, Fujie M. Urinary trypsin inhibitor (UTI) and fragments derived from UTI by limited proteolysis efficiently inhibit tumor cell invasion. Clin Exp Metastasis 1994;12:117-28.

9. Kobayashi H, Shinohara H, Takeuchi K, Itoh M, Fujie M, Saitoh M, et al. Inhibition of the soluble and the tumor cell receptor-bound plasmin by urinary trypsin inhibitor and subsequent effects on tumor cell invasion and metastasis. Can- cer Res 1994;54:844-9.

10. Hochstrasser K, Schonberger OL, Rossmanith I, Wachter E. Kunitz-type proteinase inhibitors derived by limited proteolysis of inter-~trypsin inhibitor. V. Hoppe-Seyler's Z Physiol Chem 1981;362:1357-62.

11. Wachter E, Hochstrasser K. Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-o~trypsin inhibitor. IV. Hoppe-Seyler's Z Physiol Chem 1981;362: 1351-5.

12. Albrecht GJ, Hochstrasser K, Salier J-R Elastase inhibition of the inter-mrypsin inhibitor and derived inhibitors of man mad cattle. Hoppe-Seyler's Z Physiol Chem 1983;64:1703-8.

13. Reisinger R Hochstrasser K, Albrecht GJ, Lempart K, Salier J-R Human inter-c~trypsin inhibitor: localization of the Kunitz-type domains in the N-terminal part of the molecule and their release by a trypsin-like proteinase. Biol Chem Hoppe-Seyler 1985;366:479-83.

14. Balduyck M, Laroui S, Mizon C, Mizon J. A proteoglycan related to the urinary trypsin inhibitor (UTI) links the two heavy chains of inter-~trypsin inhibitor. Biol Chem Hoppe- Seyler 1989;370:329-36.

15. Hochstrasser K, Albrecht GJ, Schonberger OL, Wachter E. Kunitz-type protease inhibitors derived by limited proteolysis of the inter-c~trypsin inhibitor. VIII. Hoppe-Seyler's Z Physiol Chem 1989;70:1689-96.

16. Gebhard W, Hochstrasser K, Fritz H, Enghild J, Pizzo SV, Salvesen G. Structure of inter-cdnhibitor: current state and proposition of a new terminology. Biol Chem Hoppe-Seyler 1990;371:13-22.

17. Salier JR Inter-c~trypsin inhibitor: emergence of a family within the Kunitz-type protease inhibitor superfamily. Trends Biochem Sci 1990;15:435-9.

18. Kobayashi H, Gotoh J, Terao T. Urinary trypsin inhibitor efficiently inhibits urokinase production in tumor necrosis factor-stimulated cells. Eur J Cell Biol 1996;71:380-6.

19. Kanayama N, el Maradny E, Halim A, Liping S, Maehara K, Kajiwara Y, et al. Urinary trypsin inhibitor prevents uterine muscle contraction by inhibition of Ca++ influx. Am J Obstet Gynecol 1995;173:192-9.

20. Kanayama N, Halim A, Maehara K, Kajiwara Y, Fujie M,

J Lab Clin Med April 1998

Terao T. Kunitz-type trypsin inhibitor prevents LPS-induced increase of cytosolic free Ca 2+ in human neutrophils and HUVEC cells. Biochem Biophys Res Commun 1995;207:324-30.

21. Kobayashi H, Ohi H, Terao T. Prevention by urinastatin of cis-diamminedichloroplatinum-induced nephrotoxicity in rabbits: comparison of urinary enzyme excretions and mor- phological alterations by electron microscopy. Asia-Oceania J Obstet Gynaecol 1991; 17:277-88.

22. Ohyama N, Hamano T, Hamakawa N, Inagaki K, Nakanishi M. Membrane fluidity and lipid hapten structure of liPo- somes affect calcium signals in antigen-specific B cells. Biochem 1991 ;30:11154-6.

23. Schootemeijer A, Van Beekhuizen AE, Tertoolen LG, De Laat SW, Akkerman JW. Cytosolic calcium ions regulate lipid mobility in the plasma membrane of the human megakaryoblastic cell line MEG-01. Eur J Biochem 1994;224:423-30.

24. Schootemeijer A, Gorter G, Tertoolen LG, De Laat SW, Akkerman JW. Relation between membrane fluidity and signal transduction in the human megakaryoblastic cell line MEG-01. Biochim Biophys Acta 1995;1236:128- 34.

25. Slater S J, Kelly MB, Taddeo FJ, Ho C. Rubin E, Stubbs CD. The modulation of protein kinase C activity by membrane lipid bilayer structure. J Biol Chem 1994;269:4866-71.

26. Wilsoncroft R Gaffen Z, Reynia S, Brain SD. The modulation by nedocromil sodium of proteases released from rat peritoneal mast cells capable of degrading vasoactive intesti- nal peptide and calcitonin gene-related peptide. Immunopharmacol 1993;25:197-204.

27. Tedeschi A, Miadonna A, Lorini M, Arquati M, Zanussi C. Receptor-operated, but not voltage-operated, calcium chan- nels are involved in basophil leucocyte activation and histamine release. Int Arch Allergy Appl Immunol 1989;90:109-11.

28. White JR, Zembryki D. Differentiation of second messenger systems in mast cell activation. Agents Actions 1989;27:410-3.

29. Emadi-Khiav B, Pearce FL Involvement of a serine protease in mast cell activation. Agents Actions 1994;41 :C37-8.

30. Picone R, Kajtaniak EL, Nielsen LS, Behrendt N, Mastroni- cola MR, Cubellis MV, et al. Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester phorbol myristate acetate. J Cell Biol 1989;108:693-702.

31. Munck Petersen C, Ejlersen E, Wendelboe Hansen R Glie- mann J. Binding of alpha-2-macroglobulin trypsin complex to human monocytes. Scand J Clin Lab Invest 1987;47:55-61.

32. Nykjaer A, Petersen CM, Christensen EI, Davidsen O, Glie- mann J. Urokinase receptors in human monocytes. Biochim Biophys Acta 1990;1052:399-407.

33. Takei M, Ueno M, Endo K, Nakagawa H. Effect of NCDC, a protease inhibitor, on histamine release from rat peritoneal mast ceils induced by anti-IgE. Biochem Biophys Res Com- mun 1991;181:1313-22.

34. White JR, Zembryki D, Hanna N, Mong S. Differential inhibition of histamine release from mast cells by protein kinase C inhibitors: staurosporine and K-252a. Biochem Pharma- col 1990;40:447-56.

35. Hayes EC, Lombardo DL, Girard Y, Maycock AL, Rokach J, Rosenthal AS, et al. Measuring leukotrienes of slow react- ing substance of anaphylaxis: development of a specific radioimmunoassay. J Immunol 1983;131:429-33.

36. Gerwins R Modification of a competitive protein binding assay for determination of inositol 1,4,5-trisphosphate. Anal Biochem 1993;210:45-9.

J Lab Clin Med Volume 131, Number 4 Kobayashi et al. 385

37. Nakashima S, Suganuma A, Matsui A, Nozawa Y. Thrombin induces a biphasic 1,2-diacylglycerol production in human platelets. Biochem J 1991;275:355-61.

38. Fujimiya H, Nakashima S, Kumada T, Nakamura Y, Miyata H, Nozawa Y. An antiallergic drug, pemirolast potassium, inhibits inositol 1,4,5-trisphosphate production and Ca 2+ mobilization in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells. Jpn J Allergol 1994;43:142-51.

39. Salier JR Martin JR Lambin R McPhee H, Hochstrasser K. Purification of the human serum inter-c~trypsin inhibitor by zinc chelate and hydrophobic interaction chromatographies. Anal Biochem 1980;109:273-83.

40. Carrero I, Rodriguez-Henche N, Guijarro LG, Recio MN, Perez-Albarsanz MA, Prieto JC. Lindane decreases forskolin-stimulated cyclic AMP accumulation but does not modify Gs in rat enterocytes. Cell Signal 1993;5:453-62.

4l. Shinitzky M, Barenholz Y. Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim Biophys Acta 1978;515:367-94.

42. Watanabe S. Inhibition of platelet aggregation by cepharan- thine is accomplished during the early, membrane-related activation process. Acta Med Okayama 1984;38:101-15.

43. Takei M, Burgoyne DL, Andersen RJ. Effect of contignas- terol on histamine release induced by anti-immunoglobulin E from rat peritoneal mast ceils. J Pharmacol Sci 1994;83:1234-5.

44. Takei M, Mitsui H, Endo K. Effect of okadaic acid on histamine release from rat peritoneal mast cells activated by anti- IgE. J Pharm Pharmacol 1993;5:750-2.

45. Licbtenstein LM. The mechanism of basophil histamine release induced by antigen and by the calcium ionophore A23187. J Immunol 1975;114:1692-9.

46. de Paulis A, Cirillo R, Ciccarelli A, de Crescenzo G, Oriente A. Characterization of the anti-inflammatory effect of FK- 506 on human mast cells. J Immunol 1991;147:4278-85.

47. Wyczolkowska J, Rydzynski K, Prouvost-Danon A. Con- canavalin A-induced activation of hamster mast cells: mor- phological changes and histamine secretion. Int Arch Aller- gy Immunol 1992;97:167-72.

48. Buhl WJ, Yu B, Gehring U. Signal transduction pathways leading to arachidonic acid release from neutrophilic HL-60 cells. The involvement of G protein, protein kinase C and phospholipase A2. Cell Signal 1995;7:351-64.

49. Himmel HM, Whorton AR, Strauss HC. Intracellular calcium, currents, and stimulus-response coupling in endothelial cells [review]. Hypertension 1993;21:112-27.

50. Fiorucci L, Erba F, Falasca L, Dini L, Ascoli E Localization and interaction of bovine pancreatic trypsin inhibitor and tryptase in the granules of bovine mast cells. Biochim Bio- phys Acta 1995;1243:407-13.

51. Hochstrasser K, Gebhard W, Albrecht G, Rasp G, Kasten- bauer E. Interaction of human mast cell tryptase and chymase with low-molecular-mass serine proteinase inhibitors from the human respiratory tract. Eur Arch Oto-Rhino- Laryngol 1993;249:455-8.

52. Itoh H, Ide H, Ishikawa N, Nawa Y. Mast cell protease inhibitor, trypstatin, is a fragment of inter-c~trypsin inhibitor light chain. J Biol Chem 1994;269:3818-22.

urinary trypsin inhibitor reduces the release of histamine from rat peritoneal mast cells

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