Arch Dermatol Res (1992) 284:8-10
.9 Springer-Verlag1992
Protein kinase C: a target for anti-inflammatory therapy? U. Amon 1 and K. R. Dietz 2
1 Department of Dermatology,Medical Universityof Ltibeck, Ratzeburger Allee 160, W-2400 Ltibeck, Federal Republic of Germany 2 Universityof Veterinary Medicine, BischoffsholerDamm 15, W-3000 Hannover, Federal Republic of Germany Received July 31, 1991/Accepted October 15, 199l
Key words: Protein kinase C -
Basophils - Skin mast
cells - Staurosporine
Skin mast cells and basophils play an important role in acute and chronic inflammatory skin diseases [5, 6]. Stimulus-secretion coupling induces the release of a number of potent proinflammatory mediators (e.g. histamine, proteases, arachidonic acid metabolites and cytokines). Recent investigations have improved our understanding of the signal transduction processes following antigen-induced activation of mast cells and basophils. It has become evident that protein kinase C (PKC), a multifunctional serine/threonine kinase, is activated after exposure to anti-IgE [9] resulting in consecutive phosphorylation of different mast cell proteins [10]. It has previously been shown that PKC can be inhibited by a number of chemically different substances in vitro [7]. Such compounds also exert a strong anti-proliferative and anti-tumour activity in vivo [4]. In the present study we investigated the effects of the strong PKC inhibitor staurosporine on the mediator release from isolated human skin mast cells and human peripheral basophils to elucidate its possible anti-allergic and anti-inflammatory efficacy in vitro. Skin mast cells were isolated enzymatically with collagenase and hyaluronidase from foreskin as previously described [1]. Basophils were obtained by venepuncture from nonallergic blood donors and isolated as previously described [9]. Cell suspensions were preincubated with the inhibitor for 15 min at 37 ~ before being challenged with an optimal dose of the stimulus for a further 30min. Histamine release was determined in both supernatants and cell pellets using an automated procedure (Technicon autoanalyser). All results are expressed as the percentage of histamine release induced by an optimal concentration of the stimulus without inhibitor and given as means
Offprint requests to." U. Amon
_+ SEM for the number of experiments noted. The histamine release occurring in the absence of any stimulus or inhibitor (spontaneous 5-10%) was substracted in all cases. PGD z and LTC 4 generation were determined in the supernatants of activated skin mast cells and basophils using commercially available radioimmunoassay systems (Amersham, Braunschweig, FRG). Staurosporine is a microbial alkaloid which inhibits isolated PKC in the nanomolar range [7]. In the present study this compound showed a strong dose-dependent inhibition of IgE-mediated histamine release from skin mast cells and basophils (Fig. 1). Maximum inhibition was 88.0 _+ 3.1% and 95.2 4-4.8%, respectively. The inhibitory effects were not dependent on the preincubation time. Very short periods (< 10s) produced similar results to a 15 min preincubation period (data not shown). When both cell populations were stimulated non-immunologically by the calcium ionophore A23187, which induces exocytosis via opening of membrane calcium channels, the inhibitory effects of staurosporine were observed only in experiments with mast cells, while the A23187-induced histamine release from basophils was virtually unaffected (Fig. 1). This suggests that the A23187-induced basophil activation may not involve PKC. Besides the exocytosis of preformed mediators, the de novo synthesis of arachidonic acid metabolites plays a crucial role in the development of cutaneous immune responses. The main metabolites generated by skin mast cells and basophils after stimulation via the Fc ~receptor are PGD 2 and LTC4, respectively. When cells were preincuabted with staurosporine for 15 rain, the antiIgE-induced generation of PGD 2 and LTC 4 was also strongly inhibited (Fig. 2). The first biochemical events induced after activation of mast cells and basophils involve the activation of phospholipase C and the subsequent hydrolysis of phosphoinositol-4,5-bisphosphate to diacylglycerol and inositol-l,4,5-trisphosphate [2]. Both second messengers are known to promote the translocation (from cytosol to membrane) and activation of PKC. The exact link between PKC activation and mediator release is still unknown. It is now clear that there are multiple sub-
100-
T
~
o
O.)
80CD
0~ r-
60.
E
c]
c-
4-0.
o cO 20, J3
j , ,
(-
I 0 5E-4
A I 1E-3
-
------A , 1E-2
A, 1E-1
. ~ J , 1
J , 10
Fig. 1. Suspensions ofbasophils (ca. 1 x 105 ceils/ ml) and skin mast cells (ca. 1 x 105 cells/ml) were preincubated with different concentrations of staurosporine for 15 min at 37 ~ before being triggered with either anti-IgE (0.01% or 10% of the anti-IgE stock solution with a titre of 1 : 8, respectively) or calcium ionophore A23187 (1 pM) for a further 30 min. Data are presented as percent inhibition of the maximum of anti-IgE or A23187induced histamine release without staurosporine. The values are given as means _+ SEM for five to seven experiments. Control histamine release (without the inhibitor) for basophils and skin mast cells was, respectively, 40.9 _+ 3.1% and 21.5 +_ 4.6% in experiments with anti-lgE, and 69.5 _+ 3.5% and 30.5 +_ 2.9% in experiments with A23187. Open circles, skin mast cells + anti-IgE; closed circles, basophils + anti-IgE; open triangles, basophils + A23187; closed triangles, skin mast cells + A23 187
Concentration of staurosporine (/~M)
1O0 9 O~ t3
"~
80-
xtC.) "O
60-
tO eq
C9
r
46 c, _0 ..i..J _ ..CI ,-tC
20-
i
o i 5E-41E-5
1E'2
1E'-1
;
1;
Fig. 2. Basophils and skin mast cells were preincubated with staurosporine and challenged with anti-IgE as described for Fig. 1. PGD 2 synthesis by skin mast cells (open circles) and LTC4 generation by basophils (closed circles) was determined in the supernatants by specific radioimmunoassays. Results are expressed as mean values _+ SEM for five experiments. PGD2 and LTC~ release without staurosporine was 21.5 + 5.3 ng/106 cells for skin mast cells and 42.3 +_ 14.1 ng/106 ceils for basophils
Concentration of staurosporine (/~M) species of PKC with different properties and probably different physiological functions [3]. PKC-mediated hyperproliferation and enhanced mediator release play a pivotal role in the pathogenesis of inflammatory skin diseases. This enzyme, therefore, seems to be a suitable target to block such deregulated mechanisms. The present results show that staurosporine has potent inhibitory effects on the IgE-mediated release of preformed and newly-generated mediators from both human skin mast cells and human peripheral basophils with low ICs0 values ( < 50 nM). Staurosporine strongly inhibits PKC in vitro (K i = 2.7 nM) [8], but is not entirely specific for this enzyme. However, it has been demonstrated recently, using T24-bladder carcinoma xenografts in athymic nude mice, that staurosporine exerts a doserelated anti-tumour activity in vivo [4]. These effects were enhanced when the staurosporine derivative CGP 41 251 with high selectivity for PKC was used [4]. Thus, development of selective PKC inhibitors may generally improve
the specific suppression of PKC-mediated immune mechanisms in vivo. In conclusion, we suggest that PKC inhibitors appear to be promising compounds in the treatment of inflammatory skin diseases. Experiments with specific PKC inhibitors are currently under way in our laboratory.
References 1. Benyon RC, Lowman MA, Church MK (1987) Human skin mast cells: their dispersion, purification, and secretory characterization. J Immunol 138:861-867 2. Church MK, Benyon RC, Clegg LS, Holgate ST (1989) Immunopharmacology of mast cells. In: Greaves MW, Shuster S (eds) Pharmacology of the skin I. Springer, Berlin Heidelberg New York Tokyo, pp 129-166 3. Farago A, NishizukaY (1990) Protein kinase C in transmembrane signalling. FEBS Lett 268:350-354
10 4. Meyer T, Regenass U, Fabro D, Alteri E, R6sel J, Miiller M, Caravatti G, Matter A (1989) A derivative of staurosporine (CGP 41 251) shows selectivity for protein kinase C inhibition and in vitro anti-proliferative as well as in vivo anti-turnout activity. Int J Cancer 43:851-856 5. Mitchell EB, Askenase PW (1983) Basophils in human disease. Clin Rev Allergy 1: 427-448 6. Rothe MJ, Nowak M, Kerdel FA (1990) The mast cell in health and disease. J Am Acad Dermatol 23:615-623 7. Tamaoki T, Nakano H (1990) Potent and specific inhibitors of protein kinase C ofmicrobial origin. Biotechnology 8 : 732 735
8. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Moromoto M, Tomita F (1986) Staurosporine, a potent inhibitor of phospholipid/Ca § § dependent protein kinase. Biochem Biophys Res Commun 135:397-402 9. Warner JA, MacGlashan DW (1989) Protein kinase C changes in human basophils. J Immunol 142:1669-1677 10. White JR, Zembryki D, Hanna N, Mong S (1990) Differential inhibition of histamine release from mast cells by protein kinase C inhibitors: staurosporine and K-252a. Biochem Pharmacol 40:447-456
.9 Springer-Verlag1992
Protein kinase C: a target for anti-inflammatory therapy? U. Amon 1 and K. R. Dietz 2
1 Department of Dermatology,Medical Universityof Ltibeck, Ratzeburger Allee 160, W-2400 Ltibeck, Federal Republic of Germany 2 Universityof Veterinary Medicine, BischoffsholerDamm 15, W-3000 Hannover, Federal Republic of Germany Received July 31, 1991/Accepted October 15, 199l
Key words: Protein kinase C -
Basophils - Skin mast
cells - Staurosporine
Skin mast cells and basophils play an important role in acute and chronic inflammatory skin diseases [5, 6]. Stimulus-secretion coupling induces the release of a number of potent proinflammatory mediators (e.g. histamine, proteases, arachidonic acid metabolites and cytokines). Recent investigations have improved our understanding of the signal transduction processes following antigen-induced activation of mast cells and basophils. It has become evident that protein kinase C (PKC), a multifunctional serine/threonine kinase, is activated after exposure to anti-IgE [9] resulting in consecutive phosphorylation of different mast cell proteins [10]. It has previously been shown that PKC can be inhibited by a number of chemically different substances in vitro [7]. Such compounds also exert a strong anti-proliferative and anti-tumour activity in vivo [4]. In the present study we investigated the effects of the strong PKC inhibitor staurosporine on the mediator release from isolated human skin mast cells and human peripheral basophils to elucidate its possible anti-allergic and anti-inflammatory efficacy in vitro. Skin mast cells were isolated enzymatically with collagenase and hyaluronidase from foreskin as previously described [1]. Basophils were obtained by venepuncture from nonallergic blood donors and isolated as previously described [9]. Cell suspensions were preincubated with the inhibitor for 15 min at 37 ~ before being challenged with an optimal dose of the stimulus for a further 30min. Histamine release was determined in both supernatants and cell pellets using an automated procedure (Technicon autoanalyser). All results are expressed as the percentage of histamine release induced by an optimal concentration of the stimulus without inhibitor and given as means
Offprint requests to." U. Amon
_+ SEM for the number of experiments noted. The histamine release occurring in the absence of any stimulus or inhibitor (spontaneous 5-10%) was substracted in all cases. PGD z and LTC 4 generation were determined in the supernatants of activated skin mast cells and basophils using commercially available radioimmunoassay systems (Amersham, Braunschweig, FRG). Staurosporine is a microbial alkaloid which inhibits isolated PKC in the nanomolar range [7]. In the present study this compound showed a strong dose-dependent inhibition of IgE-mediated histamine release from skin mast cells and basophils (Fig. 1). Maximum inhibition was 88.0 _+ 3.1% and 95.2 4-4.8%, respectively. The inhibitory effects were not dependent on the preincubation time. Very short periods (< 10s) produced similar results to a 15 min preincubation period (data not shown). When both cell populations were stimulated non-immunologically by the calcium ionophore A23187, which induces exocytosis via opening of membrane calcium channels, the inhibitory effects of staurosporine were observed only in experiments with mast cells, while the A23187-induced histamine release from basophils was virtually unaffected (Fig. 1). This suggests that the A23187-induced basophil activation may not involve PKC. Besides the exocytosis of preformed mediators, the de novo synthesis of arachidonic acid metabolites plays a crucial role in the development of cutaneous immune responses. The main metabolites generated by skin mast cells and basophils after stimulation via the Fc ~receptor are PGD 2 and LTC4, respectively. When cells were preincuabted with staurosporine for 15 rain, the antiIgE-induced generation of PGD 2 and LTC 4 was also strongly inhibited (Fig. 2). The first biochemical events induced after activation of mast cells and basophils involve the activation of phospholipase C and the subsequent hydrolysis of phosphoinositol-4,5-bisphosphate to diacylglycerol and inositol-l,4,5-trisphosphate [2]. Both second messengers are known to promote the translocation (from cytosol to membrane) and activation of PKC. The exact link between PKC activation and mediator release is still unknown. It is now clear that there are multiple sub-
100-
T
~
o
O.)
80CD
0~ r-
60.
E
c]
c-
4-0.
o cO 20, J3
j , ,
(-
I 0 5E-4
A I 1E-3
-
------A , 1E-2
A, 1E-1
. ~ J , 1
J , 10
Fig. 1. Suspensions ofbasophils (ca. 1 x 105 ceils/ ml) and skin mast cells (ca. 1 x 105 cells/ml) were preincubated with different concentrations of staurosporine for 15 min at 37 ~ before being triggered with either anti-IgE (0.01% or 10% of the anti-IgE stock solution with a titre of 1 : 8, respectively) or calcium ionophore A23187 (1 pM) for a further 30 min. Data are presented as percent inhibition of the maximum of anti-IgE or A23187induced histamine release without staurosporine. The values are given as means _+ SEM for five to seven experiments. Control histamine release (without the inhibitor) for basophils and skin mast cells was, respectively, 40.9 _+ 3.1% and 21.5 +_ 4.6% in experiments with anti-lgE, and 69.5 _+ 3.5% and 30.5 +_ 2.9% in experiments with A23187. Open circles, skin mast cells + anti-IgE; closed circles, basophils + anti-IgE; open triangles, basophils + A23187; closed triangles, skin mast cells + A23 187
Concentration of staurosporine (/~M)
1O0 9 O~ t3
"~
80-
xtC.) "O
60-
tO eq
C9
r
46 c, _0 ..i..J _ ..CI ,-tC
20-
i
o i 5E-41E-5
1E'2
1E'-1
;
1;
Fig. 2. Basophils and skin mast cells were preincubated with staurosporine and challenged with anti-IgE as described for Fig. 1. PGD 2 synthesis by skin mast cells (open circles) and LTC4 generation by basophils (closed circles) was determined in the supernatants by specific radioimmunoassays. Results are expressed as mean values _+ SEM for five experiments. PGD2 and LTC~ release without staurosporine was 21.5 + 5.3 ng/106 cells for skin mast cells and 42.3 +_ 14.1 ng/106 ceils for basophils
Concentration of staurosporine (/~M) species of PKC with different properties and probably different physiological functions [3]. PKC-mediated hyperproliferation and enhanced mediator release play a pivotal role in the pathogenesis of inflammatory skin diseases. This enzyme, therefore, seems to be a suitable target to block such deregulated mechanisms. The present results show that staurosporine has potent inhibitory effects on the IgE-mediated release of preformed and newly-generated mediators from both human skin mast cells and human peripheral basophils with low ICs0 values ( < 50 nM). Staurosporine strongly inhibits PKC in vitro (K i = 2.7 nM) [8], but is not entirely specific for this enzyme. However, it has been demonstrated recently, using T24-bladder carcinoma xenografts in athymic nude mice, that staurosporine exerts a doserelated anti-tumour activity in vivo [4]. These effects were enhanced when the staurosporine derivative CGP 41 251 with high selectivity for PKC was used [4]. Thus, development of selective PKC inhibitors may generally improve
the specific suppression of PKC-mediated immune mechanisms in vivo. In conclusion, we suggest that PKC inhibitors appear to be promising compounds in the treatment of inflammatory skin diseases. Experiments with specific PKC inhibitors are currently under way in our laboratory.
References 1. Benyon RC, Lowman MA, Church MK (1987) Human skin mast cells: their dispersion, purification, and secretory characterization. J Immunol 138:861-867 2. Church MK, Benyon RC, Clegg LS, Holgate ST (1989) Immunopharmacology of mast cells. In: Greaves MW, Shuster S (eds) Pharmacology of the skin I. Springer, Berlin Heidelberg New York Tokyo, pp 129-166 3. Farago A, NishizukaY (1990) Protein kinase C in transmembrane signalling. FEBS Lett 268:350-354
10 4. Meyer T, Regenass U, Fabro D, Alteri E, R6sel J, Miiller M, Caravatti G, Matter A (1989) A derivative of staurosporine (CGP 41 251) shows selectivity for protein kinase C inhibition and in vitro anti-proliferative as well as in vivo anti-turnout activity. Int J Cancer 43:851-856 5. Mitchell EB, Askenase PW (1983) Basophils in human disease. Clin Rev Allergy 1: 427-448 6. Rothe MJ, Nowak M, Kerdel FA (1990) The mast cell in health and disease. J Am Acad Dermatol 23:615-623 7. Tamaoki T, Nakano H (1990) Potent and specific inhibitors of protein kinase C ofmicrobial origin. Biotechnology 8 : 732 735
8. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Moromoto M, Tomita F (1986) Staurosporine, a potent inhibitor of phospholipid/Ca § § dependent protein kinase. Biochem Biophys Res Commun 135:397-402 9. Warner JA, MacGlashan DW (1989) Protein kinase C changes in human basophils. J Immunol 142:1669-1677 10. White JR, Zembryki D, Hanna N, Mong S (1990) Differential inhibition of histamine release from mast cells by protein kinase C inhibitors: staurosporine and K-252a. Biochem Pharmacol 40:447-456
