Arch Dermatol Res (1992) 284:227 231
9 Springer-Verlag1992
Etretinate or cyclosporin-A treatment normalizes the enhanced respiratory burst of polymorphonuclear leukocytes in psoriasis* R. E. Sehopf 1, J. H6eher 1, M. Rehder 1, L. Fiirber 2, and B. Morsehes 1 1 Universit/its-Hautklinik,Langenbeckstrasse1, W-6500 Mainz, Federal Republic of Germany 2 Sandoz AG, Nfirnberg, Federal Republic of Germany Received January 20, 1992
Summary. During a therapeutic trial to treat psoriasis with either etretinate or cyelosporin A (CyA) we measured the respiratory burst activity of polymorphonuelear leukocytes (PMN). Six patients received 0.5-0.75 mg/kg etretinate and 14 patients 2.5-5.0 mg/kg CyA over a period of 10 weeks. The extent of psoriasis was graded by the psoriasis area-and-severity index (PASI score). The respiratory burst of PMN isolated from the peripheral blood was measured employing luminol-enhaneed chemiluminescence at weeks 0, 3 and 10 and compared with that of 26 healthy control individuals. PMN were stimulated with zymosan particles, aggregated immunoglobulin (aggIg) and concanavalin A (ConA). Both treatment regimens improved psoriasis; at 10 weeks there was an approximate 40% PASI score reduetion under etretinate and an 80% improvement under CyA. Before treatment the respiratory burst was abnormally high under stimulation with the three stimuli in patients (p = 0.021 to < 0.0001). After 3 to 10 weeks PMN activity normalized in all patients and even tended to drop below values correlating with an improvement in skin lesions. We conclude that the elevated respiratory burst of P M N in psoriasis normalizes under treatment with both etretinate and CyA. Key words: Psoriasis - Polymorphonuclear leukocytes - Etretinate - Cyclosporin A - Chemiluminescence
Psoriasis is a neutrophilic dermatosis. In the active phase of the disease the skin is abnormally infiltrated by polymorphonuclear leukocytes (PMN) arranged as the spongiform pustules of Kogoj and Munro's microabscesses [24]. An abnormal behaviour of PMN has also been reported in the peripheral blood patients with psoriasis. While there is little doubt that PMN in patients with psoriasis are more active in terms of adherence [27, 36], chemotaxis [23, 30], enzyme contents [17, 20] and de-
granulation [5, 14, 26], controversy exists about the level of the respiratory burst of PMN in psoriasis. The term 'respiratory burst' refers to the markedly increased uptake of oxygen, the production of superoxide anion, hydrogen peroxide, hydroxyl radical, and singlet oxygen, as well as the increased activity of the hexose monophosphate shunt of PMN after appropriate stimulation [2]. The biological effects of the respiratory burst encompass the killing of microbes as well as the destruction of tissue cells including keratinocytes [2, 34]. Both normal [13, 21] and elevated levels [32] of the respiratory burst of PMN have been reported in psoriasis. In these investigations, the respiratory burst was only examined once during the course of disease in the particular patients. In addition, the extent of psoriasis was not clearly defined. Moreover, the effects of treatment on the respiratory burst have not been studied. In order to compare the respiratory burst during the course of psoriasis we examined PMN at the beginning and after three and 10 weeks of systemic treatment. We determined the extent of psoriasis employing the psoriasis area-and-severity index (PASI score) [12]. Treatment consisted ofetretinate, reported to diminish PMN chemotaxis [11, 29] as well as cyclosporin a (CyA), known to inhibit interleukin-2 (IL-2) in T lymphocytes [16]. We probed the respiratory burst of PMN using luminolenhanced chemiluminescence. We used three different stimuli for PMN: (1) zymosan particles to measure phagocytic activity, (2) aggregated immunoglobulin (aggIg) to assess Fc-receptor function and (3) concanavalin-A (ConA) to compare the activation of the respiratory burst via mannose moieties on the PMN membrane. We report that the improvement of psoriasis under treatment with either etretinate or CyA coincides with a normalization of an enhanced respiratory burst of PMN. Materials and methods Patients with psoriasis
Correspondence to: R. E. Schopf
* This paper contains data from the doctoral thesis of J. H.
For this study we recruited 20 outpatients aged 18 to 69 years with a medianage of 45 comprisingsix femalesand 14 males. All patients
228 had received no systemic therapy, including UV irradiation, for at least 3 months or antipsoriatic topical treatment for at least 1 week. The extent of skin lesions was rated using the PASI score. To enroll, patients were required to have a PASI score of > 8. Treatment regimens had been predetermined assigning in radomized fashion one-third of patients, i.e. six, to 0.5 mg/kg etretinate and two -thirds, i.e. 14, to 2.5 mg/kg CyA. The PASI was scored at weeks 0, 1, 3, 6, and 10 by the same investigator (R.E.S.). If the PASI score failed to decrease by 30% after 6 weeks, drug doses were increased to 0.75 mg/kg etretinate or 5 mg/kg CyA per day. Further details of the clinical trial are described elsewhere [35]. To measure the respiratory burst in PMN blood was drawn at weeks 0, 3 and 10.
in the C y A g r o u p h a d a P A S I score o f 12.79 _+ 3.97 (range 8.4-22.3). These values did n o t differ significantly. T o c o m p a r e the i n d i v i d u a l l y v a r y i n g P A S I scores better d u r i n g t r e a t m e n t the values are p l o t t e d in Fig. 1 o n a p e r c e n t a g e basis. F r o m these d a t a it was c o n c l u d e d that b o t h forms o f t r e a t m e n t i m p r o v e d psoriasis, a l t h o u g h C y A cleared the skin lesions m o r e r a p i d l y a n d to a greater extent.
Respiratory burst of P M N in psoriasis under treatment W h i l e the c h e m i l u m i n e s c e n c e of resting, u n s t i m u l a t e d P M N did n o t differ b e t w e e n c o n t r o l s a n d p a t i e n t s u n d e r
Control subjects A total of 26 healthy individuals comprising 10 females and 16 males aged 18 to 66 years with a median age of 40 were included.
90 in
* CyA I a Etretinate I
i
+r
~
///,/1
n=14
70
O
Measurement of the respiratory burst of P M N Luminol-enhanced chemiluminescence served to compare the respiratory burst as previously described [33] except that Dulbecco's MEM (Boehringer, Mannheim, FRG) was used as medium for short-term PMN cultures. To obtain PMN, heparinized (10 U/ml) blood diluted 1 : 4 in MEM (Gibco, Karlsruhe, FRG) was carefully layered over a Ficoll density gradient (density 1.077 g/cm 3) and centrifuged for 30 min at 400 x g to remove platelets and peripheral blood mononuclear leukocytes. The erythrocyte sediment with the buffy coat on top containing the PMN was then suspended in 5% dextran 250000 (Roth, Karlsruhe, FRG). After sedimentation for 75 min at 1 x g erythrocytes settled at the bottom of the tubes. PMN were isolated from the supernatant by washing twice in MEM. Contaminating erythrocytes were removed by incubating for 20 min at room temperature in 0.85% ammonium chloride solution (Merck, Darmstadt, FRG). After further washing in Dulbecco's MEM a cell suspension consisting of > 98% PMN with a viability of > 98% resulted as gauged by exclusion of 0.4% trypan blue (Merck). PMN (5x 105) were then incubated with the following stimuli: (1) 0.3 mg/ml zymosan particles (Sigma, St. Louis, Mo., USA) to induce phagocytosis; (2) 10 mg/ml heat-aggregated (10 rain at 62 ~ human agglg (Intraglobin, Biotest, Diagnostics, Frankfurt/Main, FRG, consisting of > 95% IgG) to stimulate via Fc-receptors; and (3) 10 gg/ml ConA (Pharmacia, Freiburg, FRG) binding specifically to mannose moieties on the outer PMN membrane. A Berthold LB 9500 (Wildbad, FRG) luminometer was used. Chemiluminescence with zymosan particles and agglg, reaching a maximum activity after about 20 to 30 min, was recorded every 10 min for 10 s over 1 h. ConA with a peak stimulation after about 3 to 5 min required recording of chemiluminescence every minute for 10 s over 10 min. Results are given as the counts of sums of the means of duplicate samples.
Statistical analyses The two-tailed student's t-test was used. Linear regression was measured by the least squares method.
Results
Efficacy of treatment A t the b e g i n n i n g o f t r e a t m e n t the six p a t i e n t s assigned to e t r e t i n a t e t r e a t m e n t exhibited a P A S I score of 15.75 _+ 3.97 ( m e a n + SD) (range 1 0 . 7 - 2 2 . 2 ) ; the 14 p a t i e n t s
to
-o
9 Springer-Verlag1992
Etretinate or cyclosporin-A treatment normalizes the enhanced respiratory burst of polymorphonuclear leukocytes in psoriasis* R. E. Sehopf 1, J. H6eher 1, M. Rehder 1, L. Fiirber 2, and B. Morsehes 1 1 Universit/its-Hautklinik,Langenbeckstrasse1, W-6500 Mainz, Federal Republic of Germany 2 Sandoz AG, Nfirnberg, Federal Republic of Germany Received January 20, 1992
Summary. During a therapeutic trial to treat psoriasis with either etretinate or cyelosporin A (CyA) we measured the respiratory burst activity of polymorphonuelear leukocytes (PMN). Six patients received 0.5-0.75 mg/kg etretinate and 14 patients 2.5-5.0 mg/kg CyA over a period of 10 weeks. The extent of psoriasis was graded by the psoriasis area-and-severity index (PASI score). The respiratory burst of PMN isolated from the peripheral blood was measured employing luminol-enhaneed chemiluminescence at weeks 0, 3 and 10 and compared with that of 26 healthy control individuals. PMN were stimulated with zymosan particles, aggregated immunoglobulin (aggIg) and concanavalin A (ConA). Both treatment regimens improved psoriasis; at 10 weeks there was an approximate 40% PASI score reduetion under etretinate and an 80% improvement under CyA. Before treatment the respiratory burst was abnormally high under stimulation with the three stimuli in patients (p = 0.021 to < 0.0001). After 3 to 10 weeks PMN activity normalized in all patients and even tended to drop below values correlating with an improvement in skin lesions. We conclude that the elevated respiratory burst of P M N in psoriasis normalizes under treatment with both etretinate and CyA. Key words: Psoriasis - Polymorphonuclear leukocytes - Etretinate - Cyclosporin A - Chemiluminescence
Psoriasis is a neutrophilic dermatosis. In the active phase of the disease the skin is abnormally infiltrated by polymorphonuclear leukocytes (PMN) arranged as the spongiform pustules of Kogoj and Munro's microabscesses [24]. An abnormal behaviour of PMN has also been reported in the peripheral blood patients with psoriasis. While there is little doubt that PMN in patients with psoriasis are more active in terms of adherence [27, 36], chemotaxis [23, 30], enzyme contents [17, 20] and de-
granulation [5, 14, 26], controversy exists about the level of the respiratory burst of PMN in psoriasis. The term 'respiratory burst' refers to the markedly increased uptake of oxygen, the production of superoxide anion, hydrogen peroxide, hydroxyl radical, and singlet oxygen, as well as the increased activity of the hexose monophosphate shunt of PMN after appropriate stimulation [2]. The biological effects of the respiratory burst encompass the killing of microbes as well as the destruction of tissue cells including keratinocytes [2, 34]. Both normal [13, 21] and elevated levels [32] of the respiratory burst of PMN have been reported in psoriasis. In these investigations, the respiratory burst was only examined once during the course of disease in the particular patients. In addition, the extent of psoriasis was not clearly defined. Moreover, the effects of treatment on the respiratory burst have not been studied. In order to compare the respiratory burst during the course of psoriasis we examined PMN at the beginning and after three and 10 weeks of systemic treatment. We determined the extent of psoriasis employing the psoriasis area-and-severity index (PASI score) [12]. Treatment consisted ofetretinate, reported to diminish PMN chemotaxis [11, 29] as well as cyclosporin a (CyA), known to inhibit interleukin-2 (IL-2) in T lymphocytes [16]. We probed the respiratory burst of PMN using luminolenhanced chemiluminescence. We used three different stimuli for PMN: (1) zymosan particles to measure phagocytic activity, (2) aggregated immunoglobulin (aggIg) to assess Fc-receptor function and (3) concanavalin-A (ConA) to compare the activation of the respiratory burst via mannose moieties on the PMN membrane. We report that the improvement of psoriasis under treatment with either etretinate or CyA coincides with a normalization of an enhanced respiratory burst of PMN. Materials and methods Patients with psoriasis
Correspondence to: R. E. Schopf
* This paper contains data from the doctoral thesis of J. H.
For this study we recruited 20 outpatients aged 18 to 69 years with a medianage of 45 comprisingsix femalesand 14 males. All patients
228 had received no systemic therapy, including UV irradiation, for at least 3 months or antipsoriatic topical treatment for at least 1 week. The extent of skin lesions was rated using the PASI score. To enroll, patients were required to have a PASI score of > 8. Treatment regimens had been predetermined assigning in radomized fashion one-third of patients, i.e. six, to 0.5 mg/kg etretinate and two -thirds, i.e. 14, to 2.5 mg/kg CyA. The PASI was scored at weeks 0, 1, 3, 6, and 10 by the same investigator (R.E.S.). If the PASI score failed to decrease by 30% after 6 weeks, drug doses were increased to 0.75 mg/kg etretinate or 5 mg/kg CyA per day. Further details of the clinical trial are described elsewhere [35]. To measure the respiratory burst in PMN blood was drawn at weeks 0, 3 and 10.
in the C y A g r o u p h a d a P A S I score o f 12.79 _+ 3.97 (range 8.4-22.3). These values did n o t differ significantly. T o c o m p a r e the i n d i v i d u a l l y v a r y i n g P A S I scores better d u r i n g t r e a t m e n t the values are p l o t t e d in Fig. 1 o n a p e r c e n t a g e basis. F r o m these d a t a it was c o n c l u d e d that b o t h forms o f t r e a t m e n t i m p r o v e d psoriasis, a l t h o u g h C y A cleared the skin lesions m o r e r a p i d l y a n d to a greater extent.
Respiratory burst of P M N in psoriasis under treatment W h i l e the c h e m i l u m i n e s c e n c e of resting, u n s t i m u l a t e d P M N did n o t differ b e t w e e n c o n t r o l s a n d p a t i e n t s u n d e r
Control subjects A total of 26 healthy individuals comprising 10 females and 16 males aged 18 to 66 years with a median age of 40 were included.
90 in
* CyA I a Etretinate I
i
+r
~
///,/1
n=14
70
O
Measurement of the respiratory burst of P M N Luminol-enhanced chemiluminescence served to compare the respiratory burst as previously described [33] except that Dulbecco's MEM (Boehringer, Mannheim, FRG) was used as medium for short-term PMN cultures. To obtain PMN, heparinized (10 U/ml) blood diluted 1 : 4 in MEM (Gibco, Karlsruhe, FRG) was carefully layered over a Ficoll density gradient (density 1.077 g/cm 3) and centrifuged for 30 min at 400 x g to remove platelets and peripheral blood mononuclear leukocytes. The erythrocyte sediment with the buffy coat on top containing the PMN was then suspended in 5% dextran 250000 (Roth, Karlsruhe, FRG). After sedimentation for 75 min at 1 x g erythrocytes settled at the bottom of the tubes. PMN were isolated from the supernatant by washing twice in MEM. Contaminating erythrocytes were removed by incubating for 20 min at room temperature in 0.85% ammonium chloride solution (Merck, Darmstadt, FRG). After further washing in Dulbecco's MEM a cell suspension consisting of > 98% PMN with a viability of > 98% resulted as gauged by exclusion of 0.4% trypan blue (Merck). PMN (5x 105) were then incubated with the following stimuli: (1) 0.3 mg/ml zymosan particles (Sigma, St. Louis, Mo., USA) to induce phagocytosis; (2) 10 mg/ml heat-aggregated (10 rain at 62 ~ human agglg (Intraglobin, Biotest, Diagnostics, Frankfurt/Main, FRG, consisting of > 95% IgG) to stimulate via Fc-receptors; and (3) 10 gg/ml ConA (Pharmacia, Freiburg, FRG) binding specifically to mannose moieties on the outer PMN membrane. A Berthold LB 9500 (Wildbad, FRG) luminometer was used. Chemiluminescence with zymosan particles and agglg, reaching a maximum activity after about 20 to 30 min, was recorded every 10 min for 10 s over 1 h. ConA with a peak stimulation after about 3 to 5 min required recording of chemiluminescence every minute for 10 s over 10 min. Results are given as the counts of sums of the means of duplicate samples.
Statistical analyses The two-tailed student's t-test was used. Linear regression was measured by the least squares method.
Results
Efficacy of treatment A t the b e g i n n i n g o f t r e a t m e n t the six p a t i e n t s assigned to e t r e t i n a t e t r e a t m e n t exhibited a P A S I score of 15.75 _+ 3.97 ( m e a n + SD) (range 1 0 . 7 - 2 2 . 2 ) ; the 14 p a t i e n t s
to
-o
