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Serum concentration and circadian profiles of cathepsins B, H and L, and their inhibitors, stefins A and B, in asthma
Clinica Chimica Acta 310 Ž2001. 113–122 www.elsevier.comrlocaterclinchim
Serum concentration and circadian profiles of cathepsins B, H and L, and their inhibitors, stefins A and B, in asthma a ˇ ˇ Nina Cimerman a,) , Pika Mesko , Stanislav Suskovic ˇ Brguljan b, Marta Krasovec ˇ ˇ b, Janko Kos a,c a
Department of Biochemical Research and Drug Design, Research and DeÕelopment DiÕision, KRKA, d.d., Cesta na Brdo 49, 1000 Ljubljana, SloÕenia b UniÕersity Clinic of Respiratory and Allergic Diseases, 4204 Golnik, SloÕenia c Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, JamoÕa 39, 1000 Ljubljana, SloÕenia Received 27 October 2000; received in revised form 10 April 2001; accepted 12 April 2001
Abstract Background: In order to determine the effect of asthma on serum concentrations of cathepsins B, H and L, and stefins A and B, the circadian and concentration profiles were followed in steroid-independent and steroid-dependent asthmatics before and after 1-week treatment with methylprednisolone and cyclosporin A. Methods: Serum samples were taken at 4-h intervals throughout a 24-h period. Cathepsin and stefin concentrations were assayed using specific ELISAs. Data were analysed by one-way ANOVA and least squares fit of 24-h cosine. Results: Temporal analysis of these proteins revealed little or no significant changes with time over a 24-h period. In comparison to normal sera, cathepsin H concentrations were elevated in all asthmatic patients, concentrations of both stefins were decreased in steroid-independent asthmatics, and stefin A concentrations were increased in steroid-dependent asthmatics before therapy. The effect of methylprednisolone treatment was demonstrated on decreased cathepsin B and increased cathepsin L concentrations in post-therapy serum samples. On the other hand, cyclosporin A treatment led to increased concentrations of cathepsins H and L. However, concentrations of stefins A and B were unaffected. Conclusions: This study associated alterations in balance of serum cysteine proteinases and their inhibitors in asthmatic patients, which has raised the possibility of their involvement in asthma pathogenesis. Validated rhythms of cathepsins and stefins in asthmatic sera exhibited temporal differences, which are too small to influence the time of sampling for their quantitative measurement over the course of a day. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Asthma; Cathepsins B, H, L; Stefins A, B; Serum; 24-h variations; Cyclosporin A; Methylprednisolone
1. Introduction
AbbreÕiations: SE, standard error; ANOVA, analysis of variance ) Corresponding author. Fax: q386-61123-3833. E-mail address: [email protected] ŽN. Cimerman..
Cysteine proteinases such as cathepsins B ŽEC 3.4.22.1., H ŽEC 3.4.22.16., and L ŽEC 3.4.22.15. constitute a major component of the lysosomal proteolytic system responsible for intracellular protein degradation and turnover w1x. They are also involved
0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 5 3 0 - 7
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in specific cellular processes including prohormone activation and antigen processing w1–3x. Cysteine proteinases are capable of degrading components of the extracellular matrix, and have been implicated in normal and pathological processes of cell growth and tissue remodelling w2–5x. By regulating cysteine proteinase activities, naturally occurring inhibitors, such as intracellular stefins and extracellular cystatins and kininogens, also play an important role in physiological and pathological processes w1,6x. Altered concentrations or activities of cysteine proteases and their inhibitors have been implicated in serious human disorders such as cancer, autoimmune diseases, kidney failure, sepsis, arthritis, Alzheimer’s disease, multiple sclerosis, epilepsy, psoriasis, osteoporosis, muscular dystrophy, inflammation, etc w1,6x. It is suggested that their serum levels may be of clinical importance for prognosis and diagnosis, especially in cancer w7–12x. Asthma is characterised by chronic airway inflammation with increased plasma exudation into the airway lumen, abnormal local secretion of proteins and airway wall remodelling. Asthmatic inflammation is still poorly defined and difficult to assess. Many inflammatory cells, mediators, and enzymes are involved, but their relative importance is not yet clear. Evidence is emerging that obstructive lung diseases such as asthma may result from a disturbed balance between proteases and their inhibitors w13x. In this report we have followed, over a 24-h period, concentration variations of cathepsins B, H and L, and their low molecular weight inhibitors stefins A and B, in sera from patients with steroid-independent and steroid-dependent asthma, before and after 1 week of treatment with methylprednisolone and cyclosporin A.
2. Materials and methods 2.1. Subjects The 21 volunteers who participated in the present study were recruited from the University Clinic of Respiratory and Allergic Diseases, Golnik. Written informed consent was obtained from all subjects. The procedures were approved and performed in
accordance with the guidelines of the regional medical ethics committee. Meals were served at 08:00, 12:30 and 18:00 h. Patients and healthy subjects were not restricted in their water intake, except for the half-hour before sampling, but were asked to abstain from other liquids and food between the meals. Lights were switched off from 22:00 to 07:00 h. Each subject was required to maintain this sleeping and waking schedule for 2 days before study. The atopic status of all subjects was defined with positive skin prick tests andror with elevated total serum IgE concentrations. All subjects had adequate diuresis, normal serum concentrations of urea, sodium, potassium, calcium and phosphorus and no pathological changes in urine proteins and urinary sediment. Nobody was hypertonic and none had any symptoms, which could suggest renal disease. Group A consisted of eight apparently healthy atopics Žmedian age, 31 years; range, 22–63 years; five females, three men. who were studied as controls. They were without asthma history and had received no medication. All asthmatic patients had a typical history of asthma and documented reversibility of forced respiratory volume in 1 s ŽFEV1 . greater than 15% after inhalation of bronchodilator. They had no acute or chronic infection, normal liver and renal function tests and they were lifelong nonsmokers. They were divided in four groups ŽB–E.. Group B consisted of eight steroid independent asthmatics Žmedian age, 50 years; range, 40–71 years; four women, four men.. They were atopics without regular use of the systemic corticosteroids. They had not received treatment with oral steroids for at least 1 month before the study or inhaled steroids for at least 3 days before the study. Group C consisted of the same eight steroid independent asthmatics after treatment with 40-mg oral methylprednisolone daily for 1 week. Group D consisted of five steroid dependent asthmatics Žmedian age, 61 years; range, 39–68 years; all women. with a continues requirement for oral corticosteroid therapy over an average period of 13 " 4 years Žmean " SE.. Dose of oral corticosteroid Žaverage daily dose of 18.6 mg, ranging from 16 to 32 mg. was not changed for at least 4 weeks before the study and remained the same through the study. Patients were also treated with inhaled beta2-
N. Cimerman et al.r Clinica Chimica Acta 310 (2001) 113–122
agonists Žsalbutamol or fenoterol. prescribed as needed and oral sustained tablets of theophylline. Serum concentrations of theophylline were within therapeutic range Ž10–20 mgrl.. Mean ŽS.E.. forced expiratory volume in 1 s ŽFEV1 . was 43% Ž17%. of the predicted value. They were all atopic. Group E consisted of the same five steroid dependent asthmatics after receiving cyclosporin A ŽSandimmunw , Sandoz Pharma, Basel. at a dose of 2 mgrkgrday for 1 week. Cyclosporin A was introduced as an alternative immunosuppressive agent.
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ŽRefs. w8,9x; Kos et al., unpublished results.. The linearity of ELISAs was tested by serial dilutions of serum samples to the levels encompassing the range of assays. A microplate reader ŽSLT Rainbow, Austria. was used to measure absorbance. The measured values of diluted samples in the ratio 1:2 were subsequently compared with the calibration curve and expressed in ngrml of serum. All determinations were performed in duplicate. The detection limits were 0.9 ngrml for cathepsin B, 2 ngrml for cathepsin H, 1.7 ngrml for cathepsin L, 0.8 ngrml for stefin A, and 0.6 ngrml for stefin B.
2.2. Samples 2.4. Statistical methods Blood was sampled on the day before and on the last day of the treatment. Blood samples of healthy subjects were collected during 1 day only. Blood was taken by venipuncture in the upright position according to National Committee for Clinical Laboratory Standards approved standard H3-A3. Samples were collected at 4-h intervals beginning at 08:00 h, with subsequent sampling at 12:00, 16:00, 20:00, 00:00, 04:00, and 08:00 h of the next day. Blood was clotted at room temperature and centrifuged subsequently at 3000 rpm. Serum was separated, aliquoted and stored frozen at y208C until analysis. 2.3. Measurement of cathepsins B, H and L, and stefins A and B Measurements of protein concentrations were done in serum samples from asthmatic patients Žgroups B–E. by commercially available specific ELISAs ŽKrka, d.d., Novo mesto, Slovenia.. The assay characteristics including recovery, within-run and between-run coefficients of variance have been determined w8,9x and the tests optimised for the use of human bodily fluids as described w10–12x. Antibodies used for cathepsin B, H and L assays recognise precursor, mature form, and enzyme–inhibitor complex. Stefin A and stefin B ELISAs preferably detect free inhibitor form besides the enzyme–inhibitor complex. Antibody specificity has been tested by immunoblotting and ELISA and cross-reactivity between the closely related cathepsins Žcathepsins B, H, L, S. and inhibitors of cysteine proteinases Žcystatin C, stefins A and B. has been excluded
All data were analysed for time effect, comparing the seven time point means by one-way ANOVA, with values in original units and also as a percentage of individual means. The latter data transformation minimised inter-individual differences. Since ANOVA can fail to obtain the actual high point of the rhythm, data were analysed individually and as a group for circadian rhythm, using single and population mean cosinor analysis involving the fit of a 24-h cosine curve by the method of least squares w14x as described previously w15–17x. The cosinor method provided both the probability of rejection of the null amplitude hypothesis for a chosen period Ž24 h in this case. and the rhythm characteristics. These included the mesor Ž24-h adjusted means., the amplitude Žhalf the difference between the maximum and the minimum fitted cosine function., and the acrophase Žtime of maximum in fitted cosine function, with midnight as the phase reference.. Group comparisons were made by the Wilcoxon matched-pairs signed-ranks test to test two related samples before and after therapy. The comparisons between two independent samples, such as healthy and asthmatic samples, were done by Mann–Whitney U–Wilcoxon rank sum W test. Two-sided P values- 0.05 were considered significant.
3. Results Tables 1–5 show the mean 24-h concentrations, the differences between the maximum and the mini-
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Table 1 Circadian characteristics for serum cathepsin B measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
A w15x Ž n s 8. B Ž n s 8. C Ž n s 8. D Ž n s 5. E Ž n s 5.
Units
ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean
24-h mean " 2 S.E.
4.5 " 0.5 19 " 2 4.5 " 0.4 63 " 10 3.9 " 0.5 74 " 21 4.0 " 0.6 24 " 5 4.1 " 0.5 28 " 8
Range
3.5–7.5 11–26 3.2–6.3 34–107 2.1–5.8 18–160 2.4–4.6 16–40 2.3–5.2 9–53
ANOVA
Least-squares fit of 24-h cosine
F
P
P
Mesor" S.E.
Amplitude
Acrophase Žh.
0.02 0.4 0.8 3.6 0.3 1.3 0.1 1.3 0.1 0.7
1.0 0.9 0.6 0.01 0.9 0.3 1.0 0.3 1.0 0.6
0.7 0.8 0.2 0.1 0.4 0.5 0.4 0.4 0.4 0.5
4.5 " 0.5 100 " 0.2 4.5 " 0.4 99 " 0.6 3.9 " 0.5 100 " 0.5 4.1 " 0.6 100 " 0.5 4.1 " 0.5 100 " 0.4
0.1 1 0.4 13 0.3 9 0.2 4 0.1 3
08:40 06:55 05:01 04:38 15:29 15:32 22:51 23:49 13:47 13:33
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference.
mum concentrations over 24 h, and circadian characteristics of cathepsins B, H and L, and stefins A and B, which were determined by specific ELISAs in sera from steroid-independent asthmatics before Žgroup B. and after Žgroup C. treatment with methylprednisolone, and in steroid-dependent asthmatics before Žgroup D. and after Žgroup E. treatment with
cyclosporin A. For comparison, the results of apparently healthy atopics Žgroup A. obtained from the previous study w15x are presented in Tables 1–5. These ELISAs have been previously used to quantify cathepsin and stefin concentrations in tissue cytosols and sera from different diseased and healthy subjects w8–12,15–17x.
Table 2 Circadian characteristics for serum cathepsin H measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
A w15x Ž n s 8. B Ž n s 8. C Ž n s 8. D Ž n s 5. E w15x Ž n s 5.
Units
ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean
24-h mean " 2 S.E.
15.0 " 2.2 112 " 51 23.1 " 3.0 73 " 9 20.9 " 1.9 65 " 13 19.2 " 1.5 52 " 8 24.5 " 2.5 45 " 10
Range
7.8–24.4 18–440 11.5–37.6 48–126 13.0–27.0 23–121 16.3–24.9 29–70 19.7–32.8 35–83
ANOVA
Least-squares fit of 24-h cosine
F
P
P
Mesor " S.E.
Amplitude
Acrophase Žh.
0.4 2.8 0.3 0.9 0.4 1.5 0.4 0.9 0.2 0.7
0.9 0.02 0.1 0.5 0.9 0.2 0.9 0.5 1.0 0.6
0.1 0.2 1.0 0.2 0.1 0.1 0.9 1.0 0.4 0.4
14.8 " 2.2 99 " 1 23.0 " 3.0 100 " 0.5 21.0 " 2 100 " 0.4 19.3 " 1.6 101 " 1 24.7 " 2.4 101 " 1
1.6 15 1.8 7 1.6 9 0.5 2 1.4 6
11:51 11:13 11:02 11:02 15:49 15:11 17:13 16:52 17:27 17:47
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference.
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Table 3 Circadian characteristics for serum cathepsin L measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
Units
A w15x Ž n s 8. ngrml % of mean B Ž n s 8. ngrml % of mean C Ž n s 8. ngrml % of mean D Ž n s 5. ngrml % of mean E Ž n s 5. ngrml % of mean
24-h mean " 2 S.E. Range
18.1 " 2.7 66 " 17 19.8 " 4.2 69 " 11 22.3 " 4.2 53 " 14 16.1 " 0.9 72 " 8 19.6 " 1.9 83 " 14
7.5–32.3 7–113 10.8–46.9 15–103 13.8–49.8 24–141 13.1–18.5 46–91 14.2–24.0 51–122
ANOVA
Least-squares fit of 24-h cosine
F
P
Mesor" S.E. Amplitude" S.E. Acrophase" S.E. Žh.
0.02 0.03 0.6 0.8 0.1 0.1 0.5 0.6 1.0 1.0
18.0 " 2.7 99 " 0.3 19.7 " 4.2 97 " 3 22.1 " 4.2 99 " 0.4 16.2 " 0.9 101 " 1 19.6 " 1.9 100 " 1
P
0.5 0.8 6.6 - 0.001 0.1 1.0 2.0 0.1 0.1 1.0 2.8 0.02 1.6 0.2 1.9 0.1 0.6 0.7 1.0 0.5
2.1 " 0.5 15 " 4 0.9 4 2.1 12 1.4 8 0.3 1
11:38 " 00:41 12:01 " 00:47 13:25 06:02 12:38 12:01 16:52 17:08 12:08 10:15
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference.
Ž P - 0.003. in all groups ŽA–E. except for cathepsin L in group D Ž P s 0.1.. This led us to perform temporal analysis not only on raw data, but also on data transformed as a percentage of the individual’s 24-h mean in order to minimise these differences between the subjects. Using single cosinor analysis significant circadian variations were found in none
Fig. 1 shows the relative concentration profiles of serum cathepsins and cystatins in healthy subjects and asthmatic patients where median concentrations in healthy sera were normalized to a relative concentration of 1.0. Inter-individual differences of all parameters were validated by ANOVA with statistical significance
Table 4 Circadian characteristics for serum stefin A measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
A w15x Ž n s 8. B Ž n s 8. C Ž n s 8. D Ž n s 5. E Ž n s 5.
Units
ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean
24-h mean " 2 S.E.
6.1 " 0.4 85 " 7 2.5 " 0.3 159 " 33 2.2 " 0.4 285 " 80 10.0 " 1.7 151 " 33 24.0 " 18.2 285 " 44
Range
4.2–8.2 32–77 1.0–4.0 10–269 1.1–3.8 34–423 5.9–16.0 46–239 4.9–96.9 160–418
ANOVA
Least-squares fit of 24-h cosine
F
P
P
Mesor " S.E.
Amplitude
Acrophase Žh.
1.0 3.1 0.5 0.7 0.6 0.6 0.3 0.6 0.1 1.5
0.4 0.01 0.8 0.7 0.9 0.8 0.9 0.7 1.0 0.2
0.2 0.2 0.3 0.3 0.5 0.6 0.6 0.6 0.3 0.5
6.1 " 0.4 100 " 1 2.6 " 0.3 99 " 1 2.4 " 0.4 120 " 16 10.0 " 1.8 99 " 2 24.2 " 18.1 103 " 2
0.3 6 0.3 13 0.4 19 1.0 9 3.9 24.4
14:26 14:34 13:00 12:43 05:13 08:34 13:23 11:19 01:05 18:06
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference.
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Table 5 Circadian characteristics for serum stefin B measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
A w15x Ž n s 8. B Ž n s 8. C Ž n s 8. D Ž n s 5. E Ž n s 5.
Units
ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean
24-h mean " 2 S.E.
3.0 " 1.2 49 " 7 0.4 " 0.1 242 " 39 0.3 " 0.1 444 " 101 2.7 " 1.9 179 " 65 2.7 " 1.8 160 " 45
Range
0.5–8.8 19–86 0.3–0.5 121–400 0.1–0.5 120–900 0.2–10.4 46–425 0.5–9.8 42–293
ANOVA
Least-squares fit of 24-h cosine
F
P
P
Mesor " S.E.
Amplitude
Acrophase Žh.
0.05 4.3 ND ND ND ND 0.02 0.9 0.2 0.9
1.0 0.002 ND ND ND ND 1.0 0.6 1.0 0.6
0.1 0.2 ND ND ND ND 0.2 0.3 0.7 0.4
3.0 " 1.2 100 " 0.3 ND ND ND ND 2.7 " 2.0 98 " 1 2.7 " 1.8 102 " 3
0.1 5 ND ND ND ND 0.4 13 0.4 14
06:52 03:46 ND ND ND ND 15:11 09:01 16:20 16:02
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference; ND, non-determinable.
or, at most, in two subjects of a separate asthmatic group for cathepsin B Žtwo subjects of group D, P - 0.03., cathepsin H Žone subject of group D, P s 0.03., cathepsin L Žtwo subjects of group C, P s 0.002; one subject of group E, P s 0.03., stefin
A Žone subject of group C, P s 0.05; one subject of group D, P s 0.05., and stefin B Žone subject of group B, P s 0.04; one subject of group D, P s 0.02; one subject of group E, P s 0.008.. The results of data analysed for time effect by one-way ANOVA
Fig. 1. Concentration profile of serum stefin A ŽSA., stefin B ŽSB., cathepsin B ŽCB., cathepsin H ŽCH. and cathepsin L ŽCL. in healthy subjects Ž n s 8., steroid-independent asthmatic patients before methylprednisolone ŽMP. treatment Ž n s 8., steroid-independent asthmatic patients after methylprednisolone treatment Ž n s 8., steroid-dependent asthmatic patients before cyclosporin A ŽCsA. treatment Ž n s 5., and in steroid-dependent asthmatic patients after cyclosporin A treatment Ž n s 5.. The median values of healthy serum concentrations were normalized to a relative concentration of 1.0.
N. Cimerman et al.r Clinica Chimica Acta 310 (2001) 113–122 Table 6 Group comparisons of serum cathepsin B ŽCB., cathepsin H ŽCH., cathepsin L ŽCL., stefin A ŽSA., and stefin B ŽSB. between group A Žeight healthy subjects., group B Žeight steroid-independent asthmatics before treatment with methylprednisolone., group C Žeight steroid-independent asthmatics after treatment with methylprednisolone., group D Žfive steroid-dependent asthmatics before treatment with cyclosporin A., and group E Žfive steroid-dependent asthmatics after treatment with cyclosporin A. a Groups CB
CH
ArB ArC ArD ArE BrC DrE BrD BrE CrD CrE
- 0.0001≠ 0.9 - 0.0001≠ 0.1 0.002≠ 0.1 - 0.0001≠ 0.4 0.07 - 0.0001≠ - 0.0001≠ 0.01≠ 0.06 0.5 0.3 0.2 0.1 0.01x 0.01≠ 0.7
0.3 0.3 0.3 0.5 0.001x 0.8 0.07 0.08 0.9 0.8
CL
SA
SB
- 0.0001x - 0.0001x 0.0002≠ 0.5 0.7 0.9 - 0.0001≠ - 0.0001≠ - 0.0001≠ - 0.0001≠
- 0.0001x - 0.0001x 0.3 0.9 0.8 0.3 - 0.0001≠ - 0.0001≠ - 0.0001≠ - 0.0001≠
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closporin A treatment, in order to determine their circadian rhythms and the effects of asthma pathogenesis and of therapy on their serum levels. The isolation of cathepsins and stefins from human organs w1x, and their detection in urine w18x and serum w15x, may indicate a passive release from cytoplasm of damaged or dying cells, or active secretion from vesicular compartment. From another group of apparently healthy subjects, we observed that beside cathepsin Brcystatin C complex there is no significant difference in stefin A concentrations between serum samples and sodium citrate plasma-matched samples, which may indicate the lack of their intracellular contribution from platelets to the measured values in healthy serum w24x. 4.1. Circadian rhythms
a
Two-tailed P values were calculated by using Wilcoxon matched-pairs signed-ranks test and Mann–Whitney U–Wilcoxon rank sum W test. Arrows indicate increase Ž≠. or decrease Žx. of the protein’s concentration compared to the first study group.
and for circadian rhythm by population mean cosinor analysis at fixed 24-h period are presented in Tables 1–5. Table 6 shows the overall differences of all serum proteins that were observed between the groups.
4. Discussion Although cathepsins B, H and L, and stefins A and B are intracellular proteins, their presence at different concentrations has been observed in several body fluids including blood w1,18x. Changed concentration profiles of cysteine proteinases and their inhibitors in blood may mirror the pathological processes where extensive proteolysis and tissue remodelling may occur, as has been already shown in cancer w7,9–12,19x, liver cirrhosis w19x, pancreatitis w20x, renal disorders w21x, autoimmune w22x and vascular w23x diseases. In this report, we measured the concentration and circadian profiles of serum cathepsins B, H and L, and stefins A and B in asthmatic patients before and after methylprednisolone or cy-
Since chronobiological features have been shown to characterise asthma, we examined circadian variations in different ways using one-way ANOVA and cosinor analysis ŽFig. 1, Tables 1–5.. ANOVA demonstrated no significant time effect in asthmatic patients except for transformed data of cathepsin B in pre-therapy samples and of cathepsin L in posttherapy samples of steroid-independent asthmatics ŽTables 1 and 3.. Using single cosinor analysis time-dependent variability was found on an individual level in a small number of asthmatic patients only. However, on a group level, validated data of all proteins determined in asthmatic sera by population mean cosinor analysis revealed no significant circadian rhythm ŽTables 1–5.. Our previous studies of concentrations of cathepsins B, H and L, cystatin C, stefins A and B in normal sera also demonstrated no apparent circadian variation, with the exception of cathepsin L where the validated rhythm exhibited a very small amplitude w15,16x. Cystatin C concentrations in asthmatic sera also showed no significant time dependence over a 24-h period w17x. It appears that the disease and type of therapy do not influence the circadian characteristics of serum cysteine proteinases and cystatins, as is the case for a respiratory function, where the circadian rhythm is more readily detected in patients than in healthy subjects, and is usually accompanied by increased amplitude with the occurrence of asthma.
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4.2. The effect of asthma Among cathepsins only cathepsin H concentrations were elevated in asthmatic sera compared to healthy controls. Concentrations of stefins A and B were decreased in steroid-independent asthmatics, whereas stefin A concentrations were increased in steroid-dependent asthmatics before therapy ŽTable 6.. The hypothesis that downregulation of an inhibitor activates a proteinase in response to different inflammatory stimuli w25,26x cannot fully account for the elevated cathepsin H, because stefins were not always decreased and cystatin C was actually increased in all asthmatic groups w17x. It is possible that increased inhibitor concentrations represent a response to the burst of proteolytic activity associated with inflammation, which is the crucial event in asthma. The number of reports following extracellular concentrations of cysteine proteinases and their inhibitors in patients with chronic lung destruction is rather limited; however, it seems that they are involved in the inflammation present in purulent bronchitis w26x, emphysema w27x, or the inflammation caused by cigarette smoking w28, 29x and hard metal particles w30x. Despite some similarities, it appears that these inflammations differ from that of asthma, with regard to the concentration profiles of cysteine proteinases and their inhibitors. When steroid-independent Žgroup B. and steroid-dependent asthmatics Žgroup D. were compared, increased concentrations of both stefins as well as cystatin C w17x were found in the latter, asthmatics having a pre-existing more severe degree of disease with a continuous requirement for corticosteroid therapy ŽTable 6.. The changes in serum concentration observed for individual cathepsins and inhibitors ŽFig. 1. suggest their different functions in the pathogenesis of asthma. 4.3. The effect of therapy After a 1-week treatment with low-dose oral methylprednisolone or cyclosporin A, a drug effect on serum cathepsin concentrations was observed, whereas no effects on serum concentrations of stefins A and B were found ŽTable 6.. However, we reported previously that methylprednisolone increased and cyclosporin A decreased serum cystatin C concentration after 1 week of therapy w17x. In this study,
methylprednisolone decreased cathepsin B concentrations, increased cathepsin L concentrations, and showed just a slight tendency to decrease cathepsin H concentrations. On the other hand, cyclosporin A left cathepsin B concentrations unchanged and increased concentrations of cathepsins H and L. In other studies, corticosteroids were reported to either reduce w31–33x, increase w33–35x or to have no effect on protein activity or concentration levels of cathepsin B w36x. It was shown that methylprednisolone and other corticosteroids may act as inhibitors of cathepsin B activity w37x. Contrary to our results, the administration of prednisolone caused no change in cathepsins L and H concentrations in rat liver w36x. Similar to our results, no effect of cyclosporin A therapy on serum cathepsin B activities were demonstrated in patients suffering from multiple sclerosis w38x. Additionally, cyclosporin A at lower doses was reported to stimulate cathepsin B, H, L q B activities in cultured proximal tubule cells, while at higher doses the effect was inhibitory w39x. Differences in the effects of methylprednisolone and cyclosporin A treatment on cathepsin and stefin concentrations may be due to different experimental conditions, such as type and duration of treatment, drug doses, different clinical features or different origin of a clinical sample.
5. Conclusion According to the temporal analysis performed by ANOVA and cosinor analysis, the time of serum sampling in asthmatic patients does not compromise the results of quantitative measurements of cathepsins B, H and L, and stefins A and B over the course of a day, regardless of whether patients were subjected to methylprednisolone or cyclosporin A therapy, supporting their usefulness for clinical evaluation as diagnostic tools. Together with our previous findings on cystatin C w17x, our results provide evidence of disturbance of the balance between cysteine proteinases and their low molecular weight inhibitors in asthmatic sera. Differentially changed concentration profiles may indicate the presence of different regulation and probably distinct roles of individual cathepsins and their inhibitors in asthma pathogenesis. However, we
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are aware that the serum concentrations may reflect both the local changes of expression and secretion of cysteine proteinases and their inhibitors, and also the systemic response to the disease. In healthy as well as in asthmatic sera, the molar concentrations of low-molecular-weight inhibitors, especially cystatin C, were significantly over the levels of their targets, cathepsins B, H and L, ensuring their effective inhibition. We can speculate that elevated concentration of cystatin C in asthmatic sera is a response to higher concentrations of cathepsin H that might participate in proteolytic events of tissue remodelling, a common feature of asthma pathogenesis.
w10x
w11x
w12x
w13x
w14x
Acknowledgements We thank Prof. Roger H. Pain for the critical reading of the manuscript. The work was supported in part by a grant from the Ministry of Science and Technology of Slovenia.
w15x
w16x
References w17x w1x Turk B, Turk V, Turk D. Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors. Biol Chem Hoppe-Seyler 1997;378:141–50. w2x Chapman HA, Riese RJ, Shi GP. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol 1997;59: 63–88. w3x Riese RJ, Chapman HA. Cathepsins and compartmentalization in antigen presentation. Curr Opin Immunol 2000;12: 107–13. w4x Reddy VY, Zhang QY, Weiss SJ. Pericellular mobilization of the tissue-destructive cysteine proteinases, cathepsins B, L, and S, by human monocyte-derived macrophages. Proc Natl Acad Sci U S A 1995;92:3849–53. w5x Shi GP, Webb AC, Foster KE, et al. Human Cathepsin S: chromosomal localization, gene structure, and tissue distribution. J Biol Chem 1994;269:11530–6. w6x Henskens YMC, Veerman ECI, Amerongen AV. Cystatins in health and disease. Biol Chem Hoppe-Seyler 1996;377: 71–86. w7x Kos J, Lah T. Cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer. Oncol Rep 1998;5:1349–61. ˇ w8x Kos J, Smid A, Krasovec M, et al. Lysosomal proteases ˇ cathepsins D, B, H, L and their inhibitors stefins A and B in head and neck cancer. Biol Chem Hoppe-Seyler 1995; 376:401–5. ˇ w9x Schweiger A, Stabuc B, Popovicˇ T, Turk V, Kos J. Enzymelinked immunosorbent assay for the detection of total cathep-
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w25x Chapman Jr. HA, Reilly Jr. JJ, Yee R, Grubb A. Identification of cystatin C, a cysteine proteinase inhibitor, as a major secretory product of human alveolar macrophages in vitro. Am Rev Respir Dis 1990;141:698–705. w26x Buttle DJ, Abrahamson M, Burnett D, et al. Human sputum cathepsin B degrades proteoglycan, is inhibited by alfa2macroglobulin and is modulated by neutrophil elastase cleavage of cathepsin B precursor and cystatin C. Biochem J 1991;276:325–31. w27x Takeyabu K, Betsuyaku T, Nishimura M, et al. Cysteine proteinases and cystatin C in bronchoalveolar lavage fluid from subjects with subclinical emphysema. Eur Respir J 1998;12:1033–9. w28x Takahashi H, Ishidoh K, Muno D, et al. Cathepsin L activity is increased in alveolar macrophages and bronchoalveolar lavage fluid of smokers. Am Rev Respir Dis 1993;147:1562– 8. w29x Warfel AH, Cardozo C, Yoo OH, Zucker-Franklin D. Cystatin C and cathepsin B production by alveolar macrophages from smokers and nonsmokers. J Leukocyte Biol 1991;49: 41–7. w30x Huaux F, Lasfargues G, Lauwerys R, Lison D. Lung toxicity of hard metal particles and production of interleukin-1, tumor necrosis factor-a, fibronectin, and cystatin C by lung phagocytes. Toxicol Appl Pharmacol 1995;132:53–62. w31x Burnett D, Stockley RA. Cathepsin B-like cysteine proteinase activity in sputum and bronchoalveolar lavage samples: relationship to inflammatory cells and effects to corticosteroids and antibiotic treatment. Clin Sci 1985;68:469–74.
w32x Crie JS, Morton P, Wildenthal K. Changes in cardiac cathepsin B activity in response to interventions that alter heart size or protein metabolism: comparison with cathepsin D. J Mol Cell Cardiol 1983;15:487–94. w33x Rajashree S, Puvanakrishnan R. Alterations in certain lysosomal glycohydrolases and cathepsins in rats on dexamethasone administration. Mol Cell Biochem 1996;154:165–70. w34x Oursler MJ, Riggs BL, Spelsberg TC. Glucocorticoid-induced activation of latent transforming growth factor-beta by normal human osteoblast-like cells. Endocrinology 1993; 133:2187–96. w35x Wang L, Lou GJ, Wang JJ, Hasselgren PO. Dexamethasone stimulates proteosome-and calcium-dependent proteolysis in cultured L6 myotubes. Shock 1998;10:298–306. w36x Inubushi T, Shikiji M, Endo K, et al. Hormonal and dietary regulation of lysosomal cysteine proteinases in liver under gluconeogenesis conditions. Biol Chem 1996;377:539–42. w37x Banik NL, Matzelle D, Terry E, Hogan EL. A new mechanism of methylprednisolone and other corticosteroids action demonstrated in vitro: inhibition of a proteinase Žcalpain. prevents myelin and cytoskeletal protein degradation. Brain Res 1997;748:205–10. w38x Olbricht CJ, Steinker M, Auch-Schwelk W, et al. Effect of cyclosporin on kidney proteolytic enzymes in men and rats. Nephrol Dial Transplant 1994;9:22–6. w39x Ling H, Vamvakas S, Schaefer L, et al. Dose-dependent stimulationrinhibition effects of cyclosporin A on lysosomal cathepsin activities in cultured proximal tubule cells. Res Exp Med ŽBerl. 1995;195:355–64.
Serum concentration and circadian profiles of cathepsins B, H and L, and their inhibitors, stefins A and B, in asthma a ˇ ˇ Nina Cimerman a,) , Pika Mesko , Stanislav Suskovic ˇ Brguljan b, Marta Krasovec ˇ ˇ b, Janko Kos a,c a
Department of Biochemical Research and Drug Design, Research and DeÕelopment DiÕision, KRKA, d.d., Cesta na Brdo 49, 1000 Ljubljana, SloÕenia b UniÕersity Clinic of Respiratory and Allergic Diseases, 4204 Golnik, SloÕenia c Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, JamoÕa 39, 1000 Ljubljana, SloÕenia Received 27 October 2000; received in revised form 10 April 2001; accepted 12 April 2001
Abstract Background: In order to determine the effect of asthma on serum concentrations of cathepsins B, H and L, and stefins A and B, the circadian and concentration profiles were followed in steroid-independent and steroid-dependent asthmatics before and after 1-week treatment with methylprednisolone and cyclosporin A. Methods: Serum samples were taken at 4-h intervals throughout a 24-h period. Cathepsin and stefin concentrations were assayed using specific ELISAs. Data were analysed by one-way ANOVA and least squares fit of 24-h cosine. Results: Temporal analysis of these proteins revealed little or no significant changes with time over a 24-h period. In comparison to normal sera, cathepsin H concentrations were elevated in all asthmatic patients, concentrations of both stefins were decreased in steroid-independent asthmatics, and stefin A concentrations were increased in steroid-dependent asthmatics before therapy. The effect of methylprednisolone treatment was demonstrated on decreased cathepsin B and increased cathepsin L concentrations in post-therapy serum samples. On the other hand, cyclosporin A treatment led to increased concentrations of cathepsins H and L. However, concentrations of stefins A and B were unaffected. Conclusions: This study associated alterations in balance of serum cysteine proteinases and their inhibitors in asthmatic patients, which has raised the possibility of their involvement in asthma pathogenesis. Validated rhythms of cathepsins and stefins in asthmatic sera exhibited temporal differences, which are too small to influence the time of sampling for their quantitative measurement over the course of a day. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Asthma; Cathepsins B, H, L; Stefins A, B; Serum; 24-h variations; Cyclosporin A; Methylprednisolone
1. Introduction
AbbreÕiations: SE, standard error; ANOVA, analysis of variance ) Corresponding author. Fax: q386-61123-3833. E-mail address: [email protected] ŽN. Cimerman..
Cysteine proteinases such as cathepsins B ŽEC 3.4.22.1., H ŽEC 3.4.22.16., and L ŽEC 3.4.22.15. constitute a major component of the lysosomal proteolytic system responsible for intracellular protein degradation and turnover w1x. They are also involved
0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 5 3 0 - 7
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in specific cellular processes including prohormone activation and antigen processing w1–3x. Cysteine proteinases are capable of degrading components of the extracellular matrix, and have been implicated in normal and pathological processes of cell growth and tissue remodelling w2–5x. By regulating cysteine proteinase activities, naturally occurring inhibitors, such as intracellular stefins and extracellular cystatins and kininogens, also play an important role in physiological and pathological processes w1,6x. Altered concentrations or activities of cysteine proteases and their inhibitors have been implicated in serious human disorders such as cancer, autoimmune diseases, kidney failure, sepsis, arthritis, Alzheimer’s disease, multiple sclerosis, epilepsy, psoriasis, osteoporosis, muscular dystrophy, inflammation, etc w1,6x. It is suggested that their serum levels may be of clinical importance for prognosis and diagnosis, especially in cancer w7–12x. Asthma is characterised by chronic airway inflammation with increased plasma exudation into the airway lumen, abnormal local secretion of proteins and airway wall remodelling. Asthmatic inflammation is still poorly defined and difficult to assess. Many inflammatory cells, mediators, and enzymes are involved, but their relative importance is not yet clear. Evidence is emerging that obstructive lung diseases such as asthma may result from a disturbed balance between proteases and their inhibitors w13x. In this report we have followed, over a 24-h period, concentration variations of cathepsins B, H and L, and their low molecular weight inhibitors stefins A and B, in sera from patients with steroid-independent and steroid-dependent asthma, before and after 1 week of treatment with methylprednisolone and cyclosporin A.
2. Materials and methods 2.1. Subjects The 21 volunteers who participated in the present study were recruited from the University Clinic of Respiratory and Allergic Diseases, Golnik. Written informed consent was obtained from all subjects. The procedures were approved and performed in
accordance with the guidelines of the regional medical ethics committee. Meals were served at 08:00, 12:30 and 18:00 h. Patients and healthy subjects were not restricted in their water intake, except for the half-hour before sampling, but were asked to abstain from other liquids and food between the meals. Lights were switched off from 22:00 to 07:00 h. Each subject was required to maintain this sleeping and waking schedule for 2 days before study. The atopic status of all subjects was defined with positive skin prick tests andror with elevated total serum IgE concentrations. All subjects had adequate diuresis, normal serum concentrations of urea, sodium, potassium, calcium and phosphorus and no pathological changes in urine proteins and urinary sediment. Nobody was hypertonic and none had any symptoms, which could suggest renal disease. Group A consisted of eight apparently healthy atopics Žmedian age, 31 years; range, 22–63 years; five females, three men. who were studied as controls. They were without asthma history and had received no medication. All asthmatic patients had a typical history of asthma and documented reversibility of forced respiratory volume in 1 s ŽFEV1 . greater than 15% after inhalation of bronchodilator. They had no acute or chronic infection, normal liver and renal function tests and they were lifelong nonsmokers. They were divided in four groups ŽB–E.. Group B consisted of eight steroid independent asthmatics Žmedian age, 50 years; range, 40–71 years; four women, four men.. They were atopics without regular use of the systemic corticosteroids. They had not received treatment with oral steroids for at least 1 month before the study or inhaled steroids for at least 3 days before the study. Group C consisted of the same eight steroid independent asthmatics after treatment with 40-mg oral methylprednisolone daily for 1 week. Group D consisted of five steroid dependent asthmatics Žmedian age, 61 years; range, 39–68 years; all women. with a continues requirement for oral corticosteroid therapy over an average period of 13 " 4 years Žmean " SE.. Dose of oral corticosteroid Žaverage daily dose of 18.6 mg, ranging from 16 to 32 mg. was not changed for at least 4 weeks before the study and remained the same through the study. Patients were also treated with inhaled beta2-
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agonists Žsalbutamol or fenoterol. prescribed as needed and oral sustained tablets of theophylline. Serum concentrations of theophylline were within therapeutic range Ž10–20 mgrl.. Mean ŽS.E.. forced expiratory volume in 1 s ŽFEV1 . was 43% Ž17%. of the predicted value. They were all atopic. Group E consisted of the same five steroid dependent asthmatics after receiving cyclosporin A ŽSandimmunw , Sandoz Pharma, Basel. at a dose of 2 mgrkgrday for 1 week. Cyclosporin A was introduced as an alternative immunosuppressive agent.
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ŽRefs. w8,9x; Kos et al., unpublished results.. The linearity of ELISAs was tested by serial dilutions of serum samples to the levels encompassing the range of assays. A microplate reader ŽSLT Rainbow, Austria. was used to measure absorbance. The measured values of diluted samples in the ratio 1:2 were subsequently compared with the calibration curve and expressed in ngrml of serum. All determinations were performed in duplicate. The detection limits were 0.9 ngrml for cathepsin B, 2 ngrml for cathepsin H, 1.7 ngrml for cathepsin L, 0.8 ngrml for stefin A, and 0.6 ngrml for stefin B.
2.2. Samples 2.4. Statistical methods Blood was sampled on the day before and on the last day of the treatment. Blood samples of healthy subjects were collected during 1 day only. Blood was taken by venipuncture in the upright position according to National Committee for Clinical Laboratory Standards approved standard H3-A3. Samples were collected at 4-h intervals beginning at 08:00 h, with subsequent sampling at 12:00, 16:00, 20:00, 00:00, 04:00, and 08:00 h of the next day. Blood was clotted at room temperature and centrifuged subsequently at 3000 rpm. Serum was separated, aliquoted and stored frozen at y208C until analysis. 2.3. Measurement of cathepsins B, H and L, and stefins A and B Measurements of protein concentrations were done in serum samples from asthmatic patients Žgroups B–E. by commercially available specific ELISAs ŽKrka, d.d., Novo mesto, Slovenia.. The assay characteristics including recovery, within-run and between-run coefficients of variance have been determined w8,9x and the tests optimised for the use of human bodily fluids as described w10–12x. Antibodies used for cathepsin B, H and L assays recognise precursor, mature form, and enzyme–inhibitor complex. Stefin A and stefin B ELISAs preferably detect free inhibitor form besides the enzyme–inhibitor complex. Antibody specificity has been tested by immunoblotting and ELISA and cross-reactivity between the closely related cathepsins Žcathepsins B, H, L, S. and inhibitors of cysteine proteinases Žcystatin C, stefins A and B. has been excluded
All data were analysed for time effect, comparing the seven time point means by one-way ANOVA, with values in original units and also as a percentage of individual means. The latter data transformation minimised inter-individual differences. Since ANOVA can fail to obtain the actual high point of the rhythm, data were analysed individually and as a group for circadian rhythm, using single and population mean cosinor analysis involving the fit of a 24-h cosine curve by the method of least squares w14x as described previously w15–17x. The cosinor method provided both the probability of rejection of the null amplitude hypothesis for a chosen period Ž24 h in this case. and the rhythm characteristics. These included the mesor Ž24-h adjusted means., the amplitude Žhalf the difference between the maximum and the minimum fitted cosine function., and the acrophase Žtime of maximum in fitted cosine function, with midnight as the phase reference.. Group comparisons were made by the Wilcoxon matched-pairs signed-ranks test to test two related samples before and after therapy. The comparisons between two independent samples, such as healthy and asthmatic samples, were done by Mann–Whitney U–Wilcoxon rank sum W test. Two-sided P values- 0.05 were considered significant.
3. Results Tables 1–5 show the mean 24-h concentrations, the differences between the maximum and the mini-
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Table 1 Circadian characteristics for serum cathepsin B measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
A w15x Ž n s 8. B Ž n s 8. C Ž n s 8. D Ž n s 5. E Ž n s 5.
Units
ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean
24-h mean " 2 S.E.
4.5 " 0.5 19 " 2 4.5 " 0.4 63 " 10 3.9 " 0.5 74 " 21 4.0 " 0.6 24 " 5 4.1 " 0.5 28 " 8
Range
3.5–7.5 11–26 3.2–6.3 34–107 2.1–5.8 18–160 2.4–4.6 16–40 2.3–5.2 9–53
ANOVA
Least-squares fit of 24-h cosine
F
P
P
Mesor" S.E.
Amplitude
Acrophase Žh.
0.02 0.4 0.8 3.6 0.3 1.3 0.1 1.3 0.1 0.7
1.0 0.9 0.6 0.01 0.9 0.3 1.0 0.3 1.0 0.6
0.7 0.8 0.2 0.1 0.4 0.5 0.4 0.4 0.4 0.5
4.5 " 0.5 100 " 0.2 4.5 " 0.4 99 " 0.6 3.9 " 0.5 100 " 0.5 4.1 " 0.6 100 " 0.5 4.1 " 0.5 100 " 0.4
0.1 1 0.4 13 0.3 9 0.2 4 0.1 3
08:40 06:55 05:01 04:38 15:29 15:32 22:51 23:49 13:47 13:33
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference.
mum concentrations over 24 h, and circadian characteristics of cathepsins B, H and L, and stefins A and B, which were determined by specific ELISAs in sera from steroid-independent asthmatics before Žgroup B. and after Žgroup C. treatment with methylprednisolone, and in steroid-dependent asthmatics before Žgroup D. and after Žgroup E. treatment with
cyclosporin A. For comparison, the results of apparently healthy atopics Žgroup A. obtained from the previous study w15x are presented in Tables 1–5. These ELISAs have been previously used to quantify cathepsin and stefin concentrations in tissue cytosols and sera from different diseased and healthy subjects w8–12,15–17x.
Table 2 Circadian characteristics for serum cathepsin H measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
A w15x Ž n s 8. B Ž n s 8. C Ž n s 8. D Ž n s 5. E w15x Ž n s 5.
Units
ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean
24-h mean " 2 S.E.
15.0 " 2.2 112 " 51 23.1 " 3.0 73 " 9 20.9 " 1.9 65 " 13 19.2 " 1.5 52 " 8 24.5 " 2.5 45 " 10
Range
7.8–24.4 18–440 11.5–37.6 48–126 13.0–27.0 23–121 16.3–24.9 29–70 19.7–32.8 35–83
ANOVA
Least-squares fit of 24-h cosine
F
P
P
Mesor " S.E.
Amplitude
Acrophase Žh.
0.4 2.8 0.3 0.9 0.4 1.5 0.4 0.9 0.2 0.7
0.9 0.02 0.1 0.5 0.9 0.2 0.9 0.5 1.0 0.6
0.1 0.2 1.0 0.2 0.1 0.1 0.9 1.0 0.4 0.4
14.8 " 2.2 99 " 1 23.0 " 3.0 100 " 0.5 21.0 " 2 100 " 0.4 19.3 " 1.6 101 " 1 24.7 " 2.4 101 " 1
1.6 15 1.8 7 1.6 9 0.5 2 1.4 6
11:51 11:13 11:02 11:02 15:49 15:11 17:13 16:52 17:27 17:47
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference.
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Table 3 Circadian characteristics for serum cathepsin L measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
Units
A w15x Ž n s 8. ngrml % of mean B Ž n s 8. ngrml % of mean C Ž n s 8. ngrml % of mean D Ž n s 5. ngrml % of mean E Ž n s 5. ngrml % of mean
24-h mean " 2 S.E. Range
18.1 " 2.7 66 " 17 19.8 " 4.2 69 " 11 22.3 " 4.2 53 " 14 16.1 " 0.9 72 " 8 19.6 " 1.9 83 " 14
7.5–32.3 7–113 10.8–46.9 15–103 13.8–49.8 24–141 13.1–18.5 46–91 14.2–24.0 51–122
ANOVA
Least-squares fit of 24-h cosine
F
P
Mesor" S.E. Amplitude" S.E. Acrophase" S.E. Žh.
0.02 0.03 0.6 0.8 0.1 0.1 0.5 0.6 1.0 1.0
18.0 " 2.7 99 " 0.3 19.7 " 4.2 97 " 3 22.1 " 4.2 99 " 0.4 16.2 " 0.9 101 " 1 19.6 " 1.9 100 " 1
P
0.5 0.8 6.6 - 0.001 0.1 1.0 2.0 0.1 0.1 1.0 2.8 0.02 1.6 0.2 1.9 0.1 0.6 0.7 1.0 0.5
2.1 " 0.5 15 " 4 0.9 4 2.1 12 1.4 8 0.3 1
11:38 " 00:41 12:01 " 00:47 13:25 06:02 12:38 12:01 16:52 17:08 12:08 10:15
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference.
Ž P - 0.003. in all groups ŽA–E. except for cathepsin L in group D Ž P s 0.1.. This led us to perform temporal analysis not only on raw data, but also on data transformed as a percentage of the individual’s 24-h mean in order to minimise these differences between the subjects. Using single cosinor analysis significant circadian variations were found in none
Fig. 1 shows the relative concentration profiles of serum cathepsins and cystatins in healthy subjects and asthmatic patients where median concentrations in healthy sera were normalized to a relative concentration of 1.0. Inter-individual differences of all parameters were validated by ANOVA with statistical significance
Table 4 Circadian characteristics for serum stefin A measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
A w15x Ž n s 8. B Ž n s 8. C Ž n s 8. D Ž n s 5. E Ž n s 5.
Units
ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean
24-h mean " 2 S.E.
6.1 " 0.4 85 " 7 2.5 " 0.3 159 " 33 2.2 " 0.4 285 " 80 10.0 " 1.7 151 " 33 24.0 " 18.2 285 " 44
Range
4.2–8.2 32–77 1.0–4.0 10–269 1.1–3.8 34–423 5.9–16.0 46–239 4.9–96.9 160–418
ANOVA
Least-squares fit of 24-h cosine
F
P
P
Mesor " S.E.
Amplitude
Acrophase Žh.
1.0 3.1 0.5 0.7 0.6 0.6 0.3 0.6 0.1 1.5
0.4 0.01 0.8 0.7 0.9 0.8 0.9 0.7 1.0 0.2
0.2 0.2 0.3 0.3 0.5 0.6 0.6 0.6 0.3 0.5
6.1 " 0.4 100 " 1 2.6 " 0.3 99 " 1 2.4 " 0.4 120 " 16 10.0 " 1.8 99 " 2 24.2 " 18.1 103 " 2
0.3 6 0.3 13 0.4 19 1.0 9 3.9 24.4
14:26 14:34 13:00 12:43 05:13 08:34 13:23 11:19 01:05 18:06
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference.
N. Cimerman et al.r Clinica Chimica Acta 310 (2001) 113–122
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Table 5 Circadian characteristics for serum stefin B measured every 4 h for 24 h in healthy subjects ŽA. w15x, steroid-independent asthmatic patients before ŽB. and after ŽC. treatment with methylprednisolone, steroid-dependent asthmatic patients before ŽD. and after ŽE. treatment with cyclosporin Aa Group
A w15x Ž n s 8. B Ž n s 8. C Ž n s 8. D Ž n s 5. E Ž n s 5.
Units
ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean ngrml % of mean
24-h mean " 2 S.E.
3.0 " 1.2 49 " 7 0.4 " 0.1 242 " 39 0.3 " 0.1 444 " 101 2.7 " 1.9 179 " 65 2.7 " 1.8 160 " 45
Range
0.5–8.8 19–86 0.3–0.5 121–400 0.1–0.5 120–900 0.2–10.4 46–425 0.5–9.8 42–293
ANOVA
Least-squares fit of 24-h cosine
F
P
P
Mesor " S.E.
Amplitude
Acrophase Žh.
0.05 4.3 ND ND ND ND 0.02 0.9 0.2 0.9
1.0 0.002 ND ND ND ND 1.0 0.6 1.0 0.6
0.1 0.2 ND ND ND ND 0.2 0.3 0.7 0.4
3.0 " 1.2 100 " 0.3 ND ND ND ND 2.7 " 2.0 98 " 1 2.7 " 1.8 102 " 3
0.1 5 ND ND ND ND 0.4 13 0.4 14
06:52 03:46 ND ND ND ND 15:11 09:01 16:20 16:02
a Statistical evaluation for circadian time effect and rhythm was determined by ANOVA and cosinor analysis. Mesor, 24-h adjusted means; Amplitude, half the difference between the maximum and the minimum fitted cosine function; % of mean, a percentage of individual seven time point means; Acrophase, time of maximum in fitted cosine function, with midnight as the phase reference; ND, non-determinable.
or, at most, in two subjects of a separate asthmatic group for cathepsin B Žtwo subjects of group D, P - 0.03., cathepsin H Žone subject of group D, P s 0.03., cathepsin L Žtwo subjects of group C, P s 0.002; one subject of group E, P s 0.03., stefin
A Žone subject of group C, P s 0.05; one subject of group D, P s 0.05., and stefin B Žone subject of group B, P s 0.04; one subject of group D, P s 0.02; one subject of group E, P s 0.008.. The results of data analysed for time effect by one-way ANOVA
Fig. 1. Concentration profile of serum stefin A ŽSA., stefin B ŽSB., cathepsin B ŽCB., cathepsin H ŽCH. and cathepsin L ŽCL. in healthy subjects Ž n s 8., steroid-independent asthmatic patients before methylprednisolone ŽMP. treatment Ž n s 8., steroid-independent asthmatic patients after methylprednisolone treatment Ž n s 8., steroid-dependent asthmatic patients before cyclosporin A ŽCsA. treatment Ž n s 5., and in steroid-dependent asthmatic patients after cyclosporin A treatment Ž n s 5.. The median values of healthy serum concentrations were normalized to a relative concentration of 1.0.
N. Cimerman et al.r Clinica Chimica Acta 310 (2001) 113–122 Table 6 Group comparisons of serum cathepsin B ŽCB., cathepsin H ŽCH., cathepsin L ŽCL., stefin A ŽSA., and stefin B ŽSB. between group A Žeight healthy subjects., group B Žeight steroid-independent asthmatics before treatment with methylprednisolone., group C Žeight steroid-independent asthmatics after treatment with methylprednisolone., group D Žfive steroid-dependent asthmatics before treatment with cyclosporin A., and group E Žfive steroid-dependent asthmatics after treatment with cyclosporin A. a Groups CB
CH
ArB ArC ArD ArE BrC DrE BrD BrE CrD CrE
- 0.0001≠ 0.9 - 0.0001≠ 0.1 0.002≠ 0.1 - 0.0001≠ 0.4 0.07 - 0.0001≠ - 0.0001≠ 0.01≠ 0.06 0.5 0.3 0.2 0.1 0.01x 0.01≠ 0.7
0.3 0.3 0.3 0.5 0.001x 0.8 0.07 0.08 0.9 0.8
CL
SA
SB
- 0.0001x - 0.0001x 0.0002≠ 0.5 0.7 0.9 - 0.0001≠ - 0.0001≠ - 0.0001≠ - 0.0001≠
- 0.0001x - 0.0001x 0.3 0.9 0.8 0.3 - 0.0001≠ - 0.0001≠ - 0.0001≠ - 0.0001≠
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closporin A treatment, in order to determine their circadian rhythms and the effects of asthma pathogenesis and of therapy on their serum levels. The isolation of cathepsins and stefins from human organs w1x, and their detection in urine w18x and serum w15x, may indicate a passive release from cytoplasm of damaged or dying cells, or active secretion from vesicular compartment. From another group of apparently healthy subjects, we observed that beside cathepsin Brcystatin C complex there is no significant difference in stefin A concentrations between serum samples and sodium citrate plasma-matched samples, which may indicate the lack of their intracellular contribution from platelets to the measured values in healthy serum w24x. 4.1. Circadian rhythms
a
Two-tailed P values were calculated by using Wilcoxon matched-pairs signed-ranks test and Mann–Whitney U–Wilcoxon rank sum W test. Arrows indicate increase Ž≠. or decrease Žx. of the protein’s concentration compared to the first study group.
and for circadian rhythm by population mean cosinor analysis at fixed 24-h period are presented in Tables 1–5. Table 6 shows the overall differences of all serum proteins that were observed between the groups.
4. Discussion Although cathepsins B, H and L, and stefins A and B are intracellular proteins, their presence at different concentrations has been observed in several body fluids including blood w1,18x. Changed concentration profiles of cysteine proteinases and their inhibitors in blood may mirror the pathological processes where extensive proteolysis and tissue remodelling may occur, as has been already shown in cancer w7,9–12,19x, liver cirrhosis w19x, pancreatitis w20x, renal disorders w21x, autoimmune w22x and vascular w23x diseases. In this report, we measured the concentration and circadian profiles of serum cathepsins B, H and L, and stefins A and B in asthmatic patients before and after methylprednisolone or cy-
Since chronobiological features have been shown to characterise asthma, we examined circadian variations in different ways using one-way ANOVA and cosinor analysis ŽFig. 1, Tables 1–5.. ANOVA demonstrated no significant time effect in asthmatic patients except for transformed data of cathepsin B in pre-therapy samples and of cathepsin L in posttherapy samples of steroid-independent asthmatics ŽTables 1 and 3.. Using single cosinor analysis time-dependent variability was found on an individual level in a small number of asthmatic patients only. However, on a group level, validated data of all proteins determined in asthmatic sera by population mean cosinor analysis revealed no significant circadian rhythm ŽTables 1–5.. Our previous studies of concentrations of cathepsins B, H and L, cystatin C, stefins A and B in normal sera also demonstrated no apparent circadian variation, with the exception of cathepsin L where the validated rhythm exhibited a very small amplitude w15,16x. Cystatin C concentrations in asthmatic sera also showed no significant time dependence over a 24-h period w17x. It appears that the disease and type of therapy do not influence the circadian characteristics of serum cysteine proteinases and cystatins, as is the case for a respiratory function, where the circadian rhythm is more readily detected in patients than in healthy subjects, and is usually accompanied by increased amplitude with the occurrence of asthma.
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4.2. The effect of asthma Among cathepsins only cathepsin H concentrations were elevated in asthmatic sera compared to healthy controls. Concentrations of stefins A and B were decreased in steroid-independent asthmatics, whereas stefin A concentrations were increased in steroid-dependent asthmatics before therapy ŽTable 6.. The hypothesis that downregulation of an inhibitor activates a proteinase in response to different inflammatory stimuli w25,26x cannot fully account for the elevated cathepsin H, because stefins were not always decreased and cystatin C was actually increased in all asthmatic groups w17x. It is possible that increased inhibitor concentrations represent a response to the burst of proteolytic activity associated with inflammation, which is the crucial event in asthma. The number of reports following extracellular concentrations of cysteine proteinases and their inhibitors in patients with chronic lung destruction is rather limited; however, it seems that they are involved in the inflammation present in purulent bronchitis w26x, emphysema w27x, or the inflammation caused by cigarette smoking w28, 29x and hard metal particles w30x. Despite some similarities, it appears that these inflammations differ from that of asthma, with regard to the concentration profiles of cysteine proteinases and their inhibitors. When steroid-independent Žgroup B. and steroid-dependent asthmatics Žgroup D. were compared, increased concentrations of both stefins as well as cystatin C w17x were found in the latter, asthmatics having a pre-existing more severe degree of disease with a continuous requirement for corticosteroid therapy ŽTable 6.. The changes in serum concentration observed for individual cathepsins and inhibitors ŽFig. 1. suggest their different functions in the pathogenesis of asthma. 4.3. The effect of therapy After a 1-week treatment with low-dose oral methylprednisolone or cyclosporin A, a drug effect on serum cathepsin concentrations was observed, whereas no effects on serum concentrations of stefins A and B were found ŽTable 6.. However, we reported previously that methylprednisolone increased and cyclosporin A decreased serum cystatin C concentration after 1 week of therapy w17x. In this study,
methylprednisolone decreased cathepsin B concentrations, increased cathepsin L concentrations, and showed just a slight tendency to decrease cathepsin H concentrations. On the other hand, cyclosporin A left cathepsin B concentrations unchanged and increased concentrations of cathepsins H and L. In other studies, corticosteroids were reported to either reduce w31–33x, increase w33–35x or to have no effect on protein activity or concentration levels of cathepsin B w36x. It was shown that methylprednisolone and other corticosteroids may act as inhibitors of cathepsin B activity w37x. Contrary to our results, the administration of prednisolone caused no change in cathepsins L and H concentrations in rat liver w36x. Similar to our results, no effect of cyclosporin A therapy on serum cathepsin B activities were demonstrated in patients suffering from multiple sclerosis w38x. Additionally, cyclosporin A at lower doses was reported to stimulate cathepsin B, H, L q B activities in cultured proximal tubule cells, while at higher doses the effect was inhibitory w39x. Differences in the effects of methylprednisolone and cyclosporin A treatment on cathepsin and stefin concentrations may be due to different experimental conditions, such as type and duration of treatment, drug doses, different clinical features or different origin of a clinical sample.
5. Conclusion According to the temporal analysis performed by ANOVA and cosinor analysis, the time of serum sampling in asthmatic patients does not compromise the results of quantitative measurements of cathepsins B, H and L, and stefins A and B over the course of a day, regardless of whether patients were subjected to methylprednisolone or cyclosporin A therapy, supporting their usefulness for clinical evaluation as diagnostic tools. Together with our previous findings on cystatin C w17x, our results provide evidence of disturbance of the balance between cysteine proteinases and their low molecular weight inhibitors in asthmatic sera. Differentially changed concentration profiles may indicate the presence of different regulation and probably distinct roles of individual cathepsins and their inhibitors in asthma pathogenesis. However, we
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are aware that the serum concentrations may reflect both the local changes of expression and secretion of cysteine proteinases and their inhibitors, and also the systemic response to the disease. In healthy as well as in asthmatic sera, the molar concentrations of low-molecular-weight inhibitors, especially cystatin C, were significantly over the levels of their targets, cathepsins B, H and L, ensuring their effective inhibition. We can speculate that elevated concentration of cystatin C in asthmatic sera is a response to higher concentrations of cathepsin H that might participate in proteolytic events of tissue remodelling, a common feature of asthma pathogenesis.
w10x
w11x
w12x
w13x
w14x
Acknowledgements We thank Prof. Roger H. Pain for the critical reading of the manuscript. The work was supported in part by a grant from the Ministry of Science and Technology of Slovenia.
w15x
w16x
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