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Equinins in equine neutrophils: Quantification in tracheobronchial secretions as an aid in the diagnosis of chronic pulmonary disease
+
The Veterinary'Journal 1998 155, 257-262
Equinins in Equine Neutrophils: Quantification in Tracheobronchial Secretions as an Aid in the Diagnosis of Chronic Pulmonary Disease A. PELLEGRINI, M. KALKINC, M. HERMANN*, B. GRI]NIG, C. WINDER and R. VON FELLENBERG Division of Applied VeterinaD, Physiology, Department of Veterinary Physiology and *Department of Veterinary Medicine, University of Zurich, Wintet~hurerstrasse260, CH-8057 Zurich, Switzerland
SUMMARY Equinins are a closely related group of proteins found in equine neutrophil granules. They demonstrate proteinase inhibiting activity restricted to microbial proteinase K and subtilisin, and they also possess antibacterial and antiviral properties. Antiproteinase K activity was measured in tracheobronchial secretions (TBS) of horses with mild (n=15), moderate (n~-30) and severe (n=16) chronic pulmonary disease, to determine its usefulness as an indicator of severity of disease and to measure neutrophil content. Determination of proteinase K inhibiting activity was based on a colorimetric assay measuring the suppression of 4-nitroaniline liberation from the synthetic substrate succinyl-~.-alanyl-alanyl-alanin-4nitroanilid, a process mediated by proteinase K. Proteinase K inhibiting activity proved to be a valid and simple indicator for assessing neutrophil content in TBS and a useful parameter to determine the severity of chronic pulmonary disease in the horse. KEVWORDS:Equinin; defensin; proteinase inhibitor; chronic pulmonary disease (CPD).
INTRODUCTION
Equinins are proteinase inhibitors, which have different electrophoretic mobilities and are located in granules of equine neutrophils (Pellegrini et aL, 1988a, 1988b; Landerer, 1993; Pellegrini, 1994). They exclusively inhibit the activity of the two microbial proteinases, proteinase K and subtilisin, and can therefore coexist with other proteinases in neutrophil granules and tracheobronchial secretions (TBS). Electrophoretically, the most cathodically migrating equinin was recently shown to be an endogenous antimicrobial agent with bactericidal activity against Streptococcus zooepidemicus, Escherichia coli and Pseudomonas aeruginosa (Couto et al., 1992, 1993) and antiviral activity against human herpes simplex virus 1, equine herpesvirus 2 and bovine parainfluenza virus 3 (St6ckli, 1993). Based on these findings, the term equinin is used here for the first time. 1090-0233/98/030257-06/$12.00/0
After the exclusion of other diseases the diagnosis of chronic pulmonary disease (CPD) relies on clinical signs, blood gas analysis and cytological evaluation of TBS. The estimated quantity of neutrophils in TBS is, however, statistically not well con'elated with the severity of the disease (Winder et al., 1989, 1990, 1991). Because of the high viscosity of TBS, the cells cannot be counted, only scored. As a result, practical problems such as reproducibility and reliability arise with subjective semiquantitive scoring. Biochemical tests that may be used in addition to neutrophil estimation, for example the measurement of proteinase activity in TBS (Grfmig et aL, 1985), have received only little attention because the quality of the enzyme substrate 'hide powder azur' is not always identical and the origin of proteinase activity is not known. The origin of equinins, however, is well documented and they can easily and specifically be measured with little interference from other proteinase inhibitors in TBS. For this purpose, the © 1998Bailli~reTindall
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THE VETERINARY JOURNAL, 155, 3
ability of proteinase K to split a synthetic, elastasespecific substrate is advantageous, because no elastase activity is measurable in equine TBS (GrOnig, 1984). The purpose of the present study was to determine whether proteinase K inhibiting activity of equinins in TBS can be used as a quantitative equivalent of neutrophil content of TBS and as indicator of the severity of CPD.
horses with dyspnoea at rest, an enlarged lung field on chest percussion and severe auscultato,3, and endoscopic abnormalities. The PaO,_, was <80 mmHg. There was a massive amount of neutrophils in TBS ( I I I I ). Some horses were so severely "affected that they were unable to work.
Tracheolnon chial secretions MATERIALS A N D M E T H O D S
Horses Sixty-one horses of various breeds were used, of which 15 had mild, 30 had moderate and 16 had severe CPD. There were five stallions, 13 mares and 43 geldings and they ranged in age fi-oln 3 to 19 ),ears (mean 8.6+3.0 years). Eighty-three per cent of the horses were over 7 years old. The clinical diagnosis of CPD was based on previously established criteria (Bracher et al., 1991) which included (1) a histoD, of decreased performance, coughing and dyspnoea; (2) exclusion of any other specific respirato D, disease; (3) elevated respirato D, rate at rest; (4) abnormal hmg sounds at rest or after injection of lobeline hydrochloride (Kantonsapotheke, CH-8057); (5) lowered PaO,_, at rest; (6) amount, colour and viscosity of TBS in the trachea; (7) estimated amount of neutrophils in smears of tracheobronchial secretions determined bv an experienced cytologist and (8) enlargement of hmg field on chest percussion. Based on these parameters the horses were allotted to the following categories: (A) Mild The group designated mild CPD, included horses with mild attscultatmT and endoscopic abnormalities; an amotmt of neutrophils in TBS scored from + to I I I I (see below); a normal PaO,_, (>90mmHg). Coughing was observed occasionally, but there was no history of performance loss. (B) Moderale The group designated moderate CPD, included horses with moderate attscultatolT and endoscopic abnormalities and accentnated abdominal breathing. The PaO._, was 80-85 m m H g and there was an increased a m o u n t of neutrophils (111) in TBS, intermittent coughing, and reduced athletic performance in some horses.
( C ) Severe The group designated severe CPD included
Tracheoscopy was carried out with a flexible fiberoptic endoscope 178.5 cm long, 14ram dialneter (Coloscope, Olympus Type LB2, Weidmann). The horses were sedated with a combination of 46 mg xylazine (Rompun, Bayer) and 5 mg 1-polamidone (Polamivet, Hoechst) per kilogram body weight intravenously. Tracheobronchial secretions were aspirated using a sterile polyethylene tube 190 cm in length and 2.7 mm in diameter through the biopsy channel of the endoscope and directed into visible secretions. Care was taken to obtain the sample quickly. A drop of TBS was placed on a glass slide, smeared, air-dried, stained with May-Grfinewald-Giemsa and examined by an experienced cytologist, who did not know the animal's history. The cells were scored fi'nm + (small amounts) to I I I I (massive amounts). The major portion of TBS was transferred to a centrithge tube and centrifi~ged at 4°C in a Sor~,all RC-5 B refrigerated superspeed centrithge (Du Pont Instruments) at 25 000 rpm for 1 11. After high-speed centrifugation, the TBS was separated into a compact sediment (gel phase), which could not be resuspended by shaking. This material was discarded. The supernatant sol phase (TBSs) was of low viscosity and varied in appearance fi'om colourless and clear to pale yellow and slightly opalescent. The relation of gel phase to sol phase varied considerably as j u d g e d visually. Since the gel phase could not be handled it was impossible to establish a quantitative relationship beuveen both components of TBS. TBSs was relnoved fi'om the centrifuge tube and fi'ozen in 0.1 mL portions at -80°(; until determination of antiproteinase K inhibitory activity.
Photometric test for proteinase K inhibiting activity of TBS Proteinase K (Boehringer, Mannheim) is a thngal proteinase (tritirachium albtun) which splits, among other substances, the synthetic elastasespecific substrate N-succi nyM.-alanyl-alanyl-alanine-p-nitroanilid [Suc-(Ala):rpNa] (Bachem, Bubendort). Tile reaction product p-nitroanilin is
EQUININS IN CHRONIC PULMONARY DISEASE
measured photometrically at 405nm by an increase in absorbency. The inhibitory activity of the equinins on the reaction can thus be determined by suppression of the absorbency increase at 405 nm.
259
1
+
a
Assay The procedure described by Geiger (1984) for pancreatic elastase inhibitors was followed with four-times reduced volumes. Twenty-five microlitres proteinase K solution (100 ~tg mL -I in 2.5 mM HCI) and 25 ~tL water (control) were mixed in a 1 mL tube. In a second tube, 25 p.L TBS~ (sample) was added to 25 ~tL proteinase K solution. To each tube 450btL buffer (0.2 M triethanolamine, pH 7.8, containing 0.03% Triton X-100 (Fluka, Buchs) was added and the tubes were equilibrated for 15 min at 25°C. Two hundred and fifty microlitres of substrate solution (1.35 mg mL -I (Suc(Ala).~-pNa) in 0.2 M triethanolamine buffer) were then added to both tubes, and the increase in absorbency at 405 nm was measured over 2 rain in a type 4010 photometer (Boehringer, Mannheim). Increased absorbency was linear in this time range. The inhibitor), activity was calculated as follows (Geiger, 1984) and expressed in international inhibitory units L-~ (IU L-~):
VL
~t
t/
where: A= change in absorbency without inhibitor Ai--change in absorbency with inhibitor VL=volume of inhibitor solution in litres t=time in minutes IU=amount of inhibitor inhibiting one unit of proteinase K.
Visualization of equinins byfibrinogen-agarose electrophoresis of TBS The procedure has been described in great detail elsewhere (Pellegrini et aL, 1984). The principle is based on the incorporation of denatured fibrinogen as proteinase substrate into agarose gel which is the carrier medium for electrophoresis. After electrophoretic resolution of equinin-containing TBS, the plate is incubated with a dilute solution of proteinase K which digests the fibrinogen with exception of the areas where equinins are located. Proteinase K is then rinsed off the plate, and the remaining fibrinogen spots, indicative of equinins, are stained with amidoblack as shown in Fig. 1.
b
C
• ,e:
:5
". ' .'t"
"
~,'z.;t 5 "
Fibrinogen-agarose electrophoresis of TBS from horses with severe CPD (a), moderate CPD (b) and mild CPD (c). The arrow indicates the site of application. Electrophoresis was carried out for 45 min at 10 V cm-L Three microlitres of TBS were applied. The bands represent undigested remains of fibrinogen at the locations where equinins inhibited proteinase K activity. Fig. 1.
Statistical analysis Statistical evaluation of the results was performed with ,a Graph Pad Inplot (Graph Pad Software Corporation, San Diego). Mean values and standard deviations were calculated. Differences between the means were evaluated by oneway analysis of variance with Bonferroni multiple comparison test. Differences were considered significant at P<0.05. RESULTS
Precision of the proteinase inhibitor assay The coefficient of variation of replicative measurements (n=12) of a pool of TBSs obtained from four horses, was 1% at a mean activity of 1475 IU L-I. The coefficient of variation of three parallel measurements (each repeated four times) of the same TBSs pool performance by three members of the laboratory was also 1%.
Proteinase-K inhibiting activity of TBSs in relation to neutrophil scores Proteinase K inhibiting activity in relation to neutrophil scores is shown in Fig. 2. The proteinase K inhibiting activity of TBS containing ++ neutrophils was significantly higher than the inhibiting activity in TBS containing + neutrophils
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T H E VETERINARY JOURNAL, 155, 3 2500
2500
2000
2000
:~
1500
D
15oo
!
1000
1000 e~
500
I
~ 500 o
Mild +
++
+++
++++
(n = 5)
( n - - 16)
(n = 26)
(n = 14)
N e u t r o p h i l score in T B S
Fig. 2. Antiproteinase K actixdty (ordinate) in relation to neutrophil scores in tracheobronchial secretions. P<0.05 between + and ++, P<0-001 between ++ and I II and P< 0.01 between I i I and I i I I. Bars: x.t.Sl).
(P<0.05). The proteinase K inhibiting activity of TBS containing I l l neutrophils was significantly higher than the inhibiting activity of TBS with ++ neutrophils (P<0.001) and the proteinase K inhibiting activity of TBS with I I II neutrophils was also significantly higher than the inhibiting activity of TBS containing I tl neutrophils (P< 0-01). The m e a n and the standard deviation values of the proteinase K inhibiting activity in relation to neutrophil score were 445_+154 IU L -~ for (+) neutrophils, 876+324 IU L -~ for (++) neutrophils, 1 2 4 4 + 3 0 6 I U L -~ for (111) neutrophils and 1565-+165 IU L -~ for (I I I f) neutrophils. In conclusion, the antiproteinase K activity in TBS reflected the estimated a m o u n t of neutrophils.
Moderate
Severe
S e v e r i t y of disease
Fig. 3. Antiproteinase K activity (ordinate) in relation to severity of disease. P<0.001 between mild and moderate, and between moderate and severe CPD. Bars: x~.sl).
Electrophoretic resolution and visualization of equinins in TBS An e l e c t r o p h e r o g r a m of equinins in TBS from horses with severe (a), m o d e r a t e (b) and mild (c) CPD is shown in Fig. 1. Four bands were visible in the TBS from horses with severe CPD. A fifth band present in extracts of neutrophil granules was not detectable in TBS. This band migrated in a slightly anodic position with respect to the application line (Pellegrini et al., 1988a). Optically it was evident that the bands were stronger in TBS of horses with severe CPD than in TBS of horses with mild CPD. In addition, the weak, most anodically migrating b a n d was only associated with severe CPD. In some horses (not shown in the figure), a doubling of the most cathodic bands was observed. Unfortunately, this m a d e their quantitative d e t e r m i n a t i o n by densitometry impossible.
Proteinase K inhibiting activity of TBS in relation to severity of CPD
DISCUSSION
In Fig. 3 the proteinase K inhibiting activity of TBS is r e p r o d u c e d for horses with mild, m o d e r a t e a n d severe CPD. With increasing severity of the disease the proteinase K inhibiting activity also increased. The difference between the three groups was highly significant (P<0.001). T h e m e a n and the standard deviation values of the proteinase K inhibiting activity of TBS in relation to severity of CPD were 600_+223 IU L -~ for horses with mild CPD, whereas for those with m o d e r a t e a n d severe CPD the mean values were 1210+ 267 IU L-' and 1598-+124 IU L -' respectively.
T h e proteinase K inhibiting activity of TBS measured with the synthetic elastase substrate Suc(Ala).~-pNa is d u e to the inhibitory effect o f the equinins, which are located in equine neutrophils (Landerer, 1993). T h e specificity of this assay for equinins is due to a combination of properties. First, the equinins have a very restricted enzyme specificity for the fungal proteinase K and the bacillar enzyme subtilisin (Pellegrini et al., 1988a, 1988b); second, proteinase K has elastolytic activity and third, TBS contains only trace a m o u n t of elastase activity (GrCmig, 1984). It was thus n o t
EQUININS IN CHRONIC PULMONARY DISEASE
surprising that proteinase K inhibiting activity was closely correlated with the microscopically determined neutrophil score of TBS and even better with the severity of CPD. The assay was easy to perform, precise and reproducible. The coefficient of variation of replicative measurements was <1% and that of parallel measurements also 1%. The test is valuable for the confirmation of visual estimations of neutrophil content when scoring cannot be performed constantly by the same experienced cytologist. It is also an indicator of the severity of CPD. It is possible that proteinase K inhibiting activity ill TBS may not only vary with neutrophil content but may also depend on the neutrophii activation level in the respiratory tract. This possibility may be further explored for diagnostic purposes. One of the main problems in CPD diagnosis is to distinguish between horses with mild CPD and healthy animals especially when TBS contains some neutrophils (Winder et al., 1989, 1990, 1991). It is possible that healthy horses may have significantly lower (or absent) proteinase inhibitory activity in TBSs than animals with mild CPD. A separate study will be required to investigate this hypothesis. At the present time, however, only animals without any detectable trace of TBS and no clinical signs can be considered to be clinically healtlay. For this reason, a control group of healthy horses was not included in this study. Although proteinase K inhibiting activity may replace neutrophil scoring, it will not eliminate qualitative cytological evaluation of TBS for the detection of disease conditions other than CPD. Detection of eosinophils is important for differential (or) additional diagnosis of parasitic bronchitis/bronchiolitis (Hermann et aL, 1988; Winder et aL, 1989). In addition, pulmonary haemorrhage may be detected (Winder, 1989) and the identification of intraphagocytic microorganisms may indicate microbial disease. Besides bronchoscopy and analysis of TBS, another method of evaluating cells in the lungs is bronchoalveolar lavage (BAL), whereby a defined amount of physiological saline is infused into the lungs through the bronchoscope wedged to a topically anaesthetized bronchus and immediately aspirated using a pump (Viel, 1983). Total cell counts are determined with the help of the recovered volume of lavage fluid. Using this method, Winder et al. (1990) found there was no significant difference between severity of disease and
261
total cell counts. Unfortunately, proteinase K inhibiting, activity cannot be measured in BAL fluid because it is too dilute. However, Winder and colleagues reported that differential cell counts yielded significantly higher relative neutrophil numbers and significantly lower relative macrophage numbers with increasing severity of disease and concluded that BAL offered no advantage over TBS sampling as a means for cytologic evaluation of CPD except in cases in which there was no TBS available in the trachea (Winder et al., 1990). Unlike collection of TBS, BAL is difficult to carry out tmder field conditions. A less complicated BAL technique introducing simply polypropylene tubing into the airways (without visual control by means of the bronchoscope and local anaesthesia of the target bronchus) has been described (Fogarty, 1990) but visual evaluation of the condition of the trachea, the bifurcatio tracheae, bronchi and the amount and consistency of TBS are clearly not possible. Measurement of proteinase K inhibiting activity of TBS relies on the availability of a diagnostic laboratory to which the material can be sent but this biochemical test is easy to perform. Thus, bronchoscopy together with TBS analysis has a number of diagnostic advantages over BAL evaluation, including the ability to measure proteinase K inhibiting activity. Equinins can be considered part of the non-oxidative defense system of equine neutrophil granules. They are probably the counterpart of defensins (Lehrer, 1993) present in humans and some other species but which could be found only in trace amounts i n equine neutrophil granules (Couto et al., 1992). Equinins are not tightly associated with granules, but leak out quite easily when granules are isolated from fractured neutrophils in vitro (Pellegrini, 1988a). This observation makes it understandable that equinins are present in significant amounts in TBS in soluble form released from stimulated neutrophils either by a secretory mechanism or following cell degeneration or death. Thus, besides intracellular microbial killing, equinins could also help to eliminate potentially pathogenic bacteria present on epithelial surfaces of the respiratory tract or other organs such as, fbr example, the female reproductive system. The electrophoretic heterogeneity of the equinins remains unexplained (Pellegrini et al., 1988a, 1988b). In TBS, the banding pattern differs from that of neuta'ophil extracts with respect to the
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THE VETERINARY.JOURNAL, 155, 3
i n t e r m e d i a r y b a n d which is slightly cathodic in TBS a n d slightly a n o d i c in n e u t r o p h i l g r a n u l a r extract. In addition, in s o m e TBS samples, we f o u n d t h r e e cathodic b a n d s instead o f two. A l t h o u g h the reason for these p h e n o m e n a r e m a i n s u n k n o w n , it may be the result o f a limited attack by p r o t e a s e s in TBS ( G r f m i g et aL, 1985) that are n o t neutralized by equinins with their restricted specificity for proteases.
REFERENCES BRACHER, V., VON FELLENBERG,R., WINDER, C. N., GRt?NI(;, G. & HEP,M.-~-NN, M. (1991). An investigation of the incidence of chronic obstructive pulmonary disease (COPD) in random populations of Swiss horses. Equine VeterinaryJournal 23, 136-41. COUTO, M. A., HARWIG,S. S. L., CULLOR,J. S., HVt;HES,J. P. & LEHRER, R. I. (1992). eNAP-2: A novel cysteinerich bactericidal peptide from equine leukocytes. Infection and hnmunity 60, 5042-7. COUTO, M. A., HARWI(;,S. S. L. & L., LEHRER,R. I. (1993). Selective inhibition of microbial serine proteases by eNAP-2, an antimicrobial peptide from equine neutrophils, hzfection and Immunity 61, 2991-4. FELLENBERG, R. (1985). Proteaseaktivitfit im Tracheobronchialsekret yon Pferden mit COPD: Pathophysiologische Bedeutung. Pferdehdlkunde 1, 55-63. FOGARTY,U. (1990). Evaluation of a bronchoalveolar lax,age technique. Equine VetednaryJourna122, 174-6. GEIGER, R. (1984). Elastases. In Methods of Enzymatic Analysis. Vol. V: Enz),mes 3: Peptidases, Proteinases and Their [nhilntors. pp. 170-6. Eds. H. U. Bergmeyer, J. Bergmeyer and M. Grassl. Weinheim: Verlag Chemie. GRONIG,G. (1984). Zur Aetiologie und Pathogenese der chronisch obstruktiven Lungenkrankheiten (COPD) des Pferdes. Dr. med. vet. dissertation, University of Z,3rich, Zftrich, Switzerl &. GRUNIG, G., HERMANN, M., Jomscrl, S., Scl-b~,REa, G. & VON FEI.t.ENBERG, R. (1985). Proteaseaktivit,ht im Tracheobronchialsekret von Pferden mit COPD: Pathophysiologische Bedeutung. Pferdeha'lkunde 1, 55-63. HER/vlANN, M., GRUNIG, G., BRACHER, V., HOWALD, B., WINDER, N. C., HORLIMANN,J. & YON FELLENBERG, R. (1988). Eosinophile Granulocyten im Tracheobronchialsekret von Pferden: Anhaltspunkt fflr eine paras-
it~.re Lungenerkrankung? SchweizerArehiv fiir Tierheib kunde 130, 19-28. L-kNDERER~ C. M. (1993). Immunocytochemischer Nachweis yon Proteinaseinhibitoren an kryofixierten nnd kryosubsfituierten neutrophilen Granulozyten und Makrophagen des Pferdes. Dr. reed. vet. dissertation, University of Zfirich, Zfirich, Switzerland. LEHRER, R. I. (1993). Defensins: Antimicrobial and cytotoxic peptides of mammalian cells. Annual Review of hnmunology I l, 105-28. PEI.LEGRINI, A. (1994). Proteinase inhibitors in animal blood with special regard to the horse: Pre-alpha-2elastase inhibitor, alpha-2-beta-l-glycoprotein and proteinase inhibitors in neutrophil granulocytes. Comparative Haematology International 4, 130-5. PELLEGRINI,A., HA(;EI.I, G. & VON FELLENBI-2RG,R. (1988a). Isolation and characterization of two new low-molecular-weight protein proteinase inhibitors from the granule-rich fraction of eqttine neutrophilic granulocytes. Biochimica Biophysica A cta 952, 309-16. PELLEGRINI,A., HAC;EI.t,G. & VON FELI.ENBERG,R. (1988b). Isolation and characterization of three protein proteinase isoinhibitors from the granular fraction of horse neutrophilic granulocytes. Biochemical Biophysical Research Communications 154, 1107-13. PEI.LE(;RINI,A., HAGEI.I,G., FRETZ, B. & YON FEI.I.ENBER(;,R. (1984). Natural proteinase inhibitors: qualitative and quantitative assay by fibrinogen-agarose electrophoresis. Analytical Biochemistry 138, 335-9. SrOc:ra.l, M. (1993). Antivirale Aktix4t yon Proteaseinhibitoren und yon Granulaextrakten der neutrophilen Granulozyten des Pferdes. Dr. med. vet. dissertation, University of Zurich, Zurich, Switzerland. VIEI., L. (1983). Structural-functional correlations of the lung in horses with small airway disease. PhD Thesis, Guelph, Ontario, Canada. WINDER, N. C., GRt?NI(;, G., HER,~IANN,M., HOWAI.D, B. & YON FELLENBFR(,,R. (1989). Comparison of respiratory secretion cytology and pulmonary histology in horses.Journal of Veterinary Medicine A 36, 32-38. WINDER, N. C., HERMANN,M., GRONIG,G., HULI.I(;ER,C. 8c YON FEI.I.ENBER(;,R. (1990). Comparison of bronchoalveolar lavage and respiratory secretion cytology in horses with clinically diagnosed chronic pulmonary disease. SchweizerArchiv ffir Tierheilkunde 132, 505-10. WINDER, N. C., GRC,NI¢;, G., HERMANN, M. & YON FELLENaER(;, R. (1991). Comparison of bronchoalveolar layage and respiratory secretion cytology in horses with histologically diagnosed pulmonary disease. Schweizer Archly flit Tierheilkunde 133, 123-30.
(Acceptedfi~rpublication 31July 1997)
The Veterinary'Journal 1998 155, 257-262
Equinins in Equine Neutrophils: Quantification in Tracheobronchial Secretions as an Aid in the Diagnosis of Chronic Pulmonary Disease A. PELLEGRINI, M. KALKINC, M. HERMANN*, B. GRI]NIG, C. WINDER and R. VON FELLENBERG Division of Applied VeterinaD, Physiology, Department of Veterinary Physiology and *Department of Veterinary Medicine, University of Zurich, Wintet~hurerstrasse260, CH-8057 Zurich, Switzerland
SUMMARY Equinins are a closely related group of proteins found in equine neutrophil granules. They demonstrate proteinase inhibiting activity restricted to microbial proteinase K and subtilisin, and they also possess antibacterial and antiviral properties. Antiproteinase K activity was measured in tracheobronchial secretions (TBS) of horses with mild (n=15), moderate (n~-30) and severe (n=16) chronic pulmonary disease, to determine its usefulness as an indicator of severity of disease and to measure neutrophil content. Determination of proteinase K inhibiting activity was based on a colorimetric assay measuring the suppression of 4-nitroaniline liberation from the synthetic substrate succinyl-~.-alanyl-alanyl-alanin-4nitroanilid, a process mediated by proteinase K. Proteinase K inhibiting activity proved to be a valid and simple indicator for assessing neutrophil content in TBS and a useful parameter to determine the severity of chronic pulmonary disease in the horse. KEVWORDS:Equinin; defensin; proteinase inhibitor; chronic pulmonary disease (CPD).
INTRODUCTION
Equinins are proteinase inhibitors, which have different electrophoretic mobilities and are located in granules of equine neutrophils (Pellegrini et aL, 1988a, 1988b; Landerer, 1993; Pellegrini, 1994). They exclusively inhibit the activity of the two microbial proteinases, proteinase K and subtilisin, and can therefore coexist with other proteinases in neutrophil granules and tracheobronchial secretions (TBS). Electrophoretically, the most cathodically migrating equinin was recently shown to be an endogenous antimicrobial agent with bactericidal activity against Streptococcus zooepidemicus, Escherichia coli and Pseudomonas aeruginosa (Couto et al., 1992, 1993) and antiviral activity against human herpes simplex virus 1, equine herpesvirus 2 and bovine parainfluenza virus 3 (St6ckli, 1993). Based on these findings, the term equinin is used here for the first time. 1090-0233/98/030257-06/$12.00/0
After the exclusion of other diseases the diagnosis of chronic pulmonary disease (CPD) relies on clinical signs, blood gas analysis and cytological evaluation of TBS. The estimated quantity of neutrophils in TBS is, however, statistically not well con'elated with the severity of the disease (Winder et al., 1989, 1990, 1991). Because of the high viscosity of TBS, the cells cannot be counted, only scored. As a result, practical problems such as reproducibility and reliability arise with subjective semiquantitive scoring. Biochemical tests that may be used in addition to neutrophil estimation, for example the measurement of proteinase activity in TBS (Grfmig et aL, 1985), have received only little attention because the quality of the enzyme substrate 'hide powder azur' is not always identical and the origin of proteinase activity is not known. The origin of equinins, however, is well documented and they can easily and specifically be measured with little interference from other proteinase inhibitors in TBS. For this purpose, the © 1998Bailli~reTindall
258
THE VETERINARY JOURNAL, 155, 3
ability of proteinase K to split a synthetic, elastasespecific substrate is advantageous, because no elastase activity is measurable in equine TBS (GrOnig, 1984). The purpose of the present study was to determine whether proteinase K inhibiting activity of equinins in TBS can be used as a quantitative equivalent of neutrophil content of TBS and as indicator of the severity of CPD.
horses with dyspnoea at rest, an enlarged lung field on chest percussion and severe auscultato,3, and endoscopic abnormalities. The PaO,_, was <80 mmHg. There was a massive amount of neutrophils in TBS ( I I I I ). Some horses were so severely "affected that they were unable to work.
Tracheolnon chial secretions MATERIALS A N D M E T H O D S
Horses Sixty-one horses of various breeds were used, of which 15 had mild, 30 had moderate and 16 had severe CPD. There were five stallions, 13 mares and 43 geldings and they ranged in age fi-oln 3 to 19 ),ears (mean 8.6+3.0 years). Eighty-three per cent of the horses were over 7 years old. The clinical diagnosis of CPD was based on previously established criteria (Bracher et al., 1991) which included (1) a histoD, of decreased performance, coughing and dyspnoea; (2) exclusion of any other specific respirato D, disease; (3) elevated respirato D, rate at rest; (4) abnormal hmg sounds at rest or after injection of lobeline hydrochloride (Kantonsapotheke, CH-8057); (5) lowered PaO,_, at rest; (6) amount, colour and viscosity of TBS in the trachea; (7) estimated amount of neutrophils in smears of tracheobronchial secretions determined bv an experienced cytologist and (8) enlargement of hmg field on chest percussion. Based on these parameters the horses were allotted to the following categories: (A) Mild The group designated mild CPD, included horses with mild attscultatmT and endoscopic abnormalities; an amotmt of neutrophils in TBS scored from + to I I I I (see below); a normal PaO,_, (>90mmHg). Coughing was observed occasionally, but there was no history of performance loss. (B) Moderale The group designated moderate CPD, included horses with moderate attscultatolT and endoscopic abnormalities and accentnated abdominal breathing. The PaO._, was 80-85 m m H g and there was an increased a m o u n t of neutrophils (111) in TBS, intermittent coughing, and reduced athletic performance in some horses.
( C ) Severe The group designated severe CPD included
Tracheoscopy was carried out with a flexible fiberoptic endoscope 178.5 cm long, 14ram dialneter (Coloscope, Olympus Type LB2, Weidmann). The horses were sedated with a combination of 46 mg xylazine (Rompun, Bayer) and 5 mg 1-polamidone (Polamivet, Hoechst) per kilogram body weight intravenously. Tracheobronchial secretions were aspirated using a sterile polyethylene tube 190 cm in length and 2.7 mm in diameter through the biopsy channel of the endoscope and directed into visible secretions. Care was taken to obtain the sample quickly. A drop of TBS was placed on a glass slide, smeared, air-dried, stained with May-Grfinewald-Giemsa and examined by an experienced cytologist, who did not know the animal's history. The cells were scored fi'nm + (small amounts) to I I I I (massive amounts). The major portion of TBS was transferred to a centrithge tube and centrifi~ged at 4°C in a Sor~,all RC-5 B refrigerated superspeed centrithge (Du Pont Instruments) at 25 000 rpm for 1 11. After high-speed centrifugation, the TBS was separated into a compact sediment (gel phase), which could not be resuspended by shaking. This material was discarded. The supernatant sol phase (TBSs) was of low viscosity and varied in appearance fi'om colourless and clear to pale yellow and slightly opalescent. The relation of gel phase to sol phase varied considerably as j u d g e d visually. Since the gel phase could not be handled it was impossible to establish a quantitative relationship beuveen both components of TBS. TBSs was relnoved fi'om the centrifuge tube and fi'ozen in 0.1 mL portions at -80°(; until determination of antiproteinase K inhibitory activity.
Photometric test for proteinase K inhibiting activity of TBS Proteinase K (Boehringer, Mannheim) is a thngal proteinase (tritirachium albtun) which splits, among other substances, the synthetic elastasespecific substrate N-succi nyM.-alanyl-alanyl-alanine-p-nitroanilid [Suc-(Ala):rpNa] (Bachem, Bubendort). Tile reaction product p-nitroanilin is
EQUININS IN CHRONIC PULMONARY DISEASE
measured photometrically at 405nm by an increase in absorbency. The inhibitory activity of the equinins on the reaction can thus be determined by suppression of the absorbency increase at 405 nm.
259
1
+
a
Assay The procedure described by Geiger (1984) for pancreatic elastase inhibitors was followed with four-times reduced volumes. Twenty-five microlitres proteinase K solution (100 ~tg mL -I in 2.5 mM HCI) and 25 ~tL water (control) were mixed in a 1 mL tube. In a second tube, 25 p.L TBS~ (sample) was added to 25 ~tL proteinase K solution. To each tube 450btL buffer (0.2 M triethanolamine, pH 7.8, containing 0.03% Triton X-100 (Fluka, Buchs) was added and the tubes were equilibrated for 15 min at 25°C. Two hundred and fifty microlitres of substrate solution (1.35 mg mL -I (Suc(Ala).~-pNa) in 0.2 M triethanolamine buffer) were then added to both tubes, and the increase in absorbency at 405 nm was measured over 2 rain in a type 4010 photometer (Boehringer, Mannheim). Increased absorbency was linear in this time range. The inhibitor), activity was calculated as follows (Geiger, 1984) and expressed in international inhibitory units L-~ (IU L-~):
VL
~t
t/
where: A= change in absorbency without inhibitor Ai--change in absorbency with inhibitor VL=volume of inhibitor solution in litres t=time in minutes IU=amount of inhibitor inhibiting one unit of proteinase K.
Visualization of equinins byfibrinogen-agarose electrophoresis of TBS The procedure has been described in great detail elsewhere (Pellegrini et aL, 1984). The principle is based on the incorporation of denatured fibrinogen as proteinase substrate into agarose gel which is the carrier medium for electrophoresis. After electrophoretic resolution of equinin-containing TBS, the plate is incubated with a dilute solution of proteinase K which digests the fibrinogen with exception of the areas where equinins are located. Proteinase K is then rinsed off the plate, and the remaining fibrinogen spots, indicative of equinins, are stained with amidoblack as shown in Fig. 1.
b
C
• ,e:
:5
". ' .'t"
"
~,'z.;t 5 "
Fibrinogen-agarose electrophoresis of TBS from horses with severe CPD (a), moderate CPD (b) and mild CPD (c). The arrow indicates the site of application. Electrophoresis was carried out for 45 min at 10 V cm-L Three microlitres of TBS were applied. The bands represent undigested remains of fibrinogen at the locations where equinins inhibited proteinase K activity. Fig. 1.
Statistical analysis Statistical evaluation of the results was performed with ,a Graph Pad Inplot (Graph Pad Software Corporation, San Diego). Mean values and standard deviations were calculated. Differences between the means were evaluated by oneway analysis of variance with Bonferroni multiple comparison test. Differences were considered significant at P<0.05. RESULTS
Precision of the proteinase inhibitor assay The coefficient of variation of replicative measurements (n=12) of a pool of TBSs obtained from four horses, was 1% at a mean activity of 1475 IU L-I. The coefficient of variation of three parallel measurements (each repeated four times) of the same TBSs pool performance by three members of the laboratory was also 1%.
Proteinase-K inhibiting activity of TBSs in relation to neutrophil scores Proteinase K inhibiting activity in relation to neutrophil scores is shown in Fig. 2. The proteinase K inhibiting activity of TBS containing ++ neutrophils was significantly higher than the inhibiting activity in TBS containing + neutrophils
260
T H E VETERINARY JOURNAL, 155, 3 2500
2500
2000
2000
:~
1500
D
15oo
!
1000
1000 e~
500
I
~ 500 o
Mild +
++
+++
++++
(n = 5)
( n - - 16)
(n = 26)
(n = 14)
N e u t r o p h i l score in T B S
Fig. 2. Antiproteinase K actixdty (ordinate) in relation to neutrophil scores in tracheobronchial secretions. P<0.05 between + and ++, P<0-001 between ++ and I II and P< 0.01 between I i I and I i I I. Bars: x.t.Sl).
(P<0.05). The proteinase K inhibiting activity of TBS containing I l l neutrophils was significantly higher than the inhibiting activity of TBS with ++ neutrophils (P<0.001) and the proteinase K inhibiting activity of TBS with I I II neutrophils was also significantly higher than the inhibiting activity of TBS containing I tl neutrophils (P< 0-01). The m e a n and the standard deviation values of the proteinase K inhibiting activity in relation to neutrophil score were 445_+154 IU L -~ for (+) neutrophils, 876+324 IU L -~ for (++) neutrophils, 1 2 4 4 + 3 0 6 I U L -~ for (111) neutrophils and 1565-+165 IU L -~ for (I I I f) neutrophils. In conclusion, the antiproteinase K activity in TBS reflected the estimated a m o u n t of neutrophils.
Moderate
Severe
S e v e r i t y of disease
Fig. 3. Antiproteinase K activity (ordinate) in relation to severity of disease. P<0.001 between mild and moderate, and between moderate and severe CPD. Bars: x~.sl).
Electrophoretic resolution and visualization of equinins in TBS An e l e c t r o p h e r o g r a m of equinins in TBS from horses with severe (a), m o d e r a t e (b) and mild (c) CPD is shown in Fig. 1. Four bands were visible in the TBS from horses with severe CPD. A fifth band present in extracts of neutrophil granules was not detectable in TBS. This band migrated in a slightly anodic position with respect to the application line (Pellegrini et al., 1988a). Optically it was evident that the bands were stronger in TBS of horses with severe CPD than in TBS of horses with mild CPD. In addition, the weak, most anodically migrating b a n d was only associated with severe CPD. In some horses (not shown in the figure), a doubling of the most cathodic bands was observed. Unfortunately, this m a d e their quantitative d e t e r m i n a t i o n by densitometry impossible.
Proteinase K inhibiting activity of TBS in relation to severity of CPD
DISCUSSION
In Fig. 3 the proteinase K inhibiting activity of TBS is r e p r o d u c e d for horses with mild, m o d e r a t e a n d severe CPD. With increasing severity of the disease the proteinase K inhibiting activity also increased. The difference between the three groups was highly significant (P<0.001). T h e m e a n and the standard deviation values of the proteinase K inhibiting activity of TBS in relation to severity of CPD were 600_+223 IU L -~ for horses with mild CPD, whereas for those with m o d e r a t e a n d severe CPD the mean values were 1210+ 267 IU L-' and 1598-+124 IU L -' respectively.
T h e proteinase K inhibiting activity of TBS measured with the synthetic elastase substrate Suc(Ala).~-pNa is d u e to the inhibitory effect o f the equinins, which are located in equine neutrophils (Landerer, 1993). T h e specificity of this assay for equinins is due to a combination of properties. First, the equinins have a very restricted enzyme specificity for the fungal proteinase K and the bacillar enzyme subtilisin (Pellegrini et al., 1988a, 1988b); second, proteinase K has elastolytic activity and third, TBS contains only trace a m o u n t of elastase activity (GrCmig, 1984). It was thus n o t
EQUININS IN CHRONIC PULMONARY DISEASE
surprising that proteinase K inhibiting activity was closely correlated with the microscopically determined neutrophil score of TBS and even better with the severity of CPD. The assay was easy to perform, precise and reproducible. The coefficient of variation of replicative measurements was <1% and that of parallel measurements also 1%. The test is valuable for the confirmation of visual estimations of neutrophil content when scoring cannot be performed constantly by the same experienced cytologist. It is also an indicator of the severity of CPD. It is possible that proteinase K inhibiting activity ill TBS may not only vary with neutrophil content but may also depend on the neutrophii activation level in the respiratory tract. This possibility may be further explored for diagnostic purposes. One of the main problems in CPD diagnosis is to distinguish between horses with mild CPD and healthy animals especially when TBS contains some neutrophils (Winder et al., 1989, 1990, 1991). It is possible that healthy horses may have significantly lower (or absent) proteinase inhibitory activity in TBSs than animals with mild CPD. A separate study will be required to investigate this hypothesis. At the present time, however, only animals without any detectable trace of TBS and no clinical signs can be considered to be clinically healtlay. For this reason, a control group of healthy horses was not included in this study. Although proteinase K inhibiting activity may replace neutrophil scoring, it will not eliminate qualitative cytological evaluation of TBS for the detection of disease conditions other than CPD. Detection of eosinophils is important for differential (or) additional diagnosis of parasitic bronchitis/bronchiolitis (Hermann et aL, 1988; Winder et aL, 1989). In addition, pulmonary haemorrhage may be detected (Winder, 1989) and the identification of intraphagocytic microorganisms may indicate microbial disease. Besides bronchoscopy and analysis of TBS, another method of evaluating cells in the lungs is bronchoalveolar lavage (BAL), whereby a defined amount of physiological saline is infused into the lungs through the bronchoscope wedged to a topically anaesthetized bronchus and immediately aspirated using a pump (Viel, 1983). Total cell counts are determined with the help of the recovered volume of lavage fluid. Using this method, Winder et al. (1990) found there was no significant difference between severity of disease and
261
total cell counts. Unfortunately, proteinase K inhibiting, activity cannot be measured in BAL fluid because it is too dilute. However, Winder and colleagues reported that differential cell counts yielded significantly higher relative neutrophil numbers and significantly lower relative macrophage numbers with increasing severity of disease and concluded that BAL offered no advantage over TBS sampling as a means for cytologic evaluation of CPD except in cases in which there was no TBS available in the trachea (Winder et al., 1990). Unlike collection of TBS, BAL is difficult to carry out tmder field conditions. A less complicated BAL technique introducing simply polypropylene tubing into the airways (without visual control by means of the bronchoscope and local anaesthesia of the target bronchus) has been described (Fogarty, 1990) but visual evaluation of the condition of the trachea, the bifurcatio tracheae, bronchi and the amount and consistency of TBS are clearly not possible. Measurement of proteinase K inhibiting activity of TBS relies on the availability of a diagnostic laboratory to which the material can be sent but this biochemical test is easy to perform. Thus, bronchoscopy together with TBS analysis has a number of diagnostic advantages over BAL evaluation, including the ability to measure proteinase K inhibiting activity. Equinins can be considered part of the non-oxidative defense system of equine neutrophil granules. They are probably the counterpart of defensins (Lehrer, 1993) present in humans and some other species but which could be found only in trace amounts i n equine neutrophil granules (Couto et al., 1992). Equinins are not tightly associated with granules, but leak out quite easily when granules are isolated from fractured neutrophils in vitro (Pellegrini, 1988a). This observation makes it understandable that equinins are present in significant amounts in TBS in soluble form released from stimulated neutrophils either by a secretory mechanism or following cell degeneration or death. Thus, besides intracellular microbial killing, equinins could also help to eliminate potentially pathogenic bacteria present on epithelial surfaces of the respiratory tract or other organs such as, fbr example, the female reproductive system. The electrophoretic heterogeneity of the equinins remains unexplained (Pellegrini et al., 1988a, 1988b). In TBS, the banding pattern differs from that of neuta'ophil extracts with respect to the
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THE VETERINARY.JOURNAL, 155, 3
i n t e r m e d i a r y b a n d which is slightly cathodic in TBS a n d slightly a n o d i c in n e u t r o p h i l g r a n u l a r extract. In addition, in s o m e TBS samples, we f o u n d t h r e e cathodic b a n d s instead o f two. A l t h o u g h the reason for these p h e n o m e n a r e m a i n s u n k n o w n , it may be the result o f a limited attack by p r o t e a s e s in TBS ( G r f m i g et aL, 1985) that are n o t neutralized by equinins with their restricted specificity for proteases.
REFERENCES BRACHER, V., VON FELLENBERG,R., WINDER, C. N., GRt?NI(;, G. & HEP,M.-~-NN, M. (1991). An investigation of the incidence of chronic obstructive pulmonary disease (COPD) in random populations of Swiss horses. Equine VeterinaryJournal 23, 136-41. COUTO, M. A., HARWIG,S. S. L., CULLOR,J. S., HVt;HES,J. P. & LEHRER, R. I. (1992). eNAP-2: A novel cysteinerich bactericidal peptide from equine leukocytes. Infection and hnmunity 60, 5042-7. COUTO, M. A., HARWI(;,S. S. L. & L., LEHRER,R. I. (1993). Selective inhibition of microbial serine proteases by eNAP-2, an antimicrobial peptide from equine neutrophils, hzfection and Immunity 61, 2991-4. FELLENBERG, R. (1985). Proteaseaktivitfit im Tracheobronchialsekret yon Pferden mit COPD: Pathophysiologische Bedeutung. Pferdehdlkunde 1, 55-63. FOGARTY,U. (1990). Evaluation of a bronchoalveolar lax,age technique. Equine VetednaryJourna122, 174-6. GEIGER, R. (1984). Elastases. In Methods of Enzymatic Analysis. Vol. V: Enz),mes 3: Peptidases, Proteinases and Their [nhilntors. pp. 170-6. Eds. H. U. Bergmeyer, J. Bergmeyer and M. Grassl. Weinheim: Verlag Chemie. GRONIG,G. (1984). Zur Aetiologie und Pathogenese der chronisch obstruktiven Lungenkrankheiten (COPD) des Pferdes. Dr. med. vet. dissertation, University of Z,3rich, Zftrich, Switzerl &. GRUNIG, G., HERMANN, M., Jomscrl, S., Scl-b~,REa, G. & VON FEI.t.ENBERG, R. (1985). Proteaseaktivit,ht im Tracheobronchialsekret von Pferden mit COPD: Pathophysiologische Bedeutung. Pferdeha'lkunde 1, 55-63. HER/vlANN, M., GRUNIG, G., BRACHER, V., HOWALD, B., WINDER, N. C., HORLIMANN,J. & YON FELLENBERG, R. (1988). Eosinophile Granulocyten im Tracheobronchialsekret von Pferden: Anhaltspunkt fflr eine paras-
it~.re Lungenerkrankung? SchweizerArehiv fiir Tierheib kunde 130, 19-28. L-kNDERER~ C. M. (1993). Immunocytochemischer Nachweis yon Proteinaseinhibitoren an kryofixierten nnd kryosubsfituierten neutrophilen Granulozyten und Makrophagen des Pferdes. Dr. reed. vet. dissertation, University of Zfirich, Zfirich, Switzerland. LEHRER, R. I. (1993). Defensins: Antimicrobial and cytotoxic peptides of mammalian cells. Annual Review of hnmunology I l, 105-28. PEI.LEGRINI, A. (1994). Proteinase inhibitors in animal blood with special regard to the horse: Pre-alpha-2elastase inhibitor, alpha-2-beta-l-glycoprotein and proteinase inhibitors in neutrophil granulocytes. Comparative Haematology International 4, 130-5. PELLEGRINI,A., HA(;EI.I, G. & VON FELLENBI-2RG,R. (1988a). Isolation and characterization of two new low-molecular-weight protein proteinase inhibitors from the granule-rich fraction of eqttine neutrophilic granulocytes. Biochimica Biophysica A cta 952, 309-16. PELLEGRINI,A., HAC;EI.t,G. & VON FELI.ENBERG,R. (1988b). Isolation and characterization of three protein proteinase isoinhibitors from the granular fraction of horse neutrophilic granulocytes. Biochemical Biophysical Research Communications 154, 1107-13. PEI.LE(;RINI,A., HAGEI.I,G., FRETZ, B. & YON FEI.I.ENBER(;,R. (1984). Natural proteinase inhibitors: qualitative and quantitative assay by fibrinogen-agarose electrophoresis. Analytical Biochemistry 138, 335-9. SrOc:ra.l, M. (1993). Antivirale Aktix4t yon Proteaseinhibitoren und yon Granulaextrakten der neutrophilen Granulozyten des Pferdes. Dr. med. vet. dissertation, University of Zurich, Zurich, Switzerland. VIEI., L. (1983). Structural-functional correlations of the lung in horses with small airway disease. PhD Thesis, Guelph, Ontario, Canada. WINDER, N. C., GRt?NI(;, G., HER,~IANN,M., HOWAI.D, B. & YON FELLENBFR(,,R. (1989). Comparison of respiratory secretion cytology and pulmonary histology in horses.Journal of Veterinary Medicine A 36, 32-38. WINDER, N. C., HERMANN,M., GRONIG,G., HULI.I(;ER,C. 8c YON FEI.I.ENBER(;,R. (1990). Comparison of bronchoalveolar lavage and respiratory secretion cytology in horses with clinically diagnosed chronic pulmonary disease. SchweizerArchiv ffir Tierheilkunde 132, 505-10. WINDER, N. C., GRC,NI¢;, G., HERMANN, M. & YON FELLENaER(;, R. (1991). Comparison of bronchoalveolar layage and respiratory secretion cytology in horses with histologically diagnosed pulmonary disease. Schweizer Archly flit Tierheilkunde 133, 123-30.
(Acceptedfi~rpublication 31July 1997)