fluorescent bands under UV light (366 nm). Materials. RPMI 1640 medium, penicillin, and streptomy- cin were obtained from GIBCO BRL, Paisley, Renfrewshire,.
INFECTION AND IMMUNITY, Oct. 1991, p. 3581-3588
Vol. 59, No. 10
0019-9567/91/103581-08$02.00/0 Copyright © 1991, American Society for Microbiology
Alterations in Cysteine Proteinase Content of Rat Lung Associated with Development of Pneumocystis carinii Infection DAVID J. HAYES,* COLIN R. STUBBERFIELD, JEFFREY D. McBRIDE, AND DIANE L. WILSON
Biochemical Sciences, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, United Kingdom Received 30 April 1991/Accepted 13 July 1991
The rate of hydrolysis of three cysteine-type proteinase substrates, N-benzyloxycarbonyl-Arg-Arg-4-methyl7-coumarylamide (AMC) (cathepsin B), Arg-AMC (cathepsin H), and N-benzyloxycarbonyl-Phe-Arg-AMC (cathepsin L), were determined in rat lung throughout the time course of the induction of Pneumocystis carinii infection by immunosuppression. Cathepsin B-like and cathepsin L-like activities fell below control values initially, but from week 8 of the immunosuppressive treatment significant increases above the control were noted. Cathepsin H-like activity was greater than control levels from week 3, and by week 12 it was 7,600% of the mean control value. When compared with the relative degree of infection, as assessed from the number of cysts present in lung impression smears, cathepsin B-like and cathepsin L-like activities were significantly increased only at heavy parasite burdens while cathepsin H-like activity displayed a close correlation with parasite number (r = 0.884; P < 0.001). Activity was detected in lysates of purified P. carinii with all three substrates. Treatment of heavily infected animals with co-trimoxazole cleared the lungs of P. carinii, and this was accompanied by a marked reduction in proteinase activity, in particular, cathepsin H-like activity, which fell from 108- to 3-fold the mean control value following drug treatment. Analysis of cathepsin H isozyme patterns by fluorography following isoelectric focusing revealed differences between treated and control lung samples. In the immunosuppressed group, there was a time-dependent increase in the intensity of some of the bands observed in the controls and an appearance of several novel bands which corresponded to bands observed in lysates of P. carinii. It is likely, therefore, that the increased proteinase activity observed in the treated group is due, at least in part, to isozymes from P. carinii; consequently, cathepsin H-like activity might be of use diagnostically in the identification of P. carinii infection and in the estimation of parasite burden.
Pneumocystis carinii is a pathogen of the lung which can cause a life-threatening pneumonia in immunocompromised individuals if untreated (9, 13, 15, 17). Historically, it occurred in malnourished infants and individuals with congenital defects of the immune system or in those on immunosuppressive therapy (cancer and organ transplant patients) (9, 13, 18). However, with the appearance of AIDS, a new, highly susceptible population has been recognized (18, 23, 25, 27). Indeed, P. carinii-induced pneumonia is a major complication in AIDS patients, and its management is hampered by a greatly increased incidence and severity of adverse reactions to standard therapies (18, 23). The need for new treatments for P. carinii pneumonia has stimulated interest in the organism, but knowledge of its biochemistry, biology, and epidemiology is still poor. There has been increasing recognition of a causal relationship between the pathogenicity of a number of lung disorders and disturbances in the regulation of proteinase activities (42). The best-described example of this is the relationship between neutrophil elastase activity and an inherited deficiency of a1-antitrypsin which causes emphysema at an early age (42). In this condition, elastase activity outstrips the ability of the endogenous proteinase inhibitors present in the lung (termed the proteinase screen), causing a cascading cycle of lung cell destruction (42). With this background in mind, we undertook to examine proteinase activity in P. carinii-infected lungs from immunocompromised rats. Increases in the activity of cysteine proteinases have also been reported in other lung disease states (16, 26, 37). Cathepsins *
B and L are highly active, broad-acting proteases which together with cathepsin H are mechanistically related to the much studied plant enzyme papain (2). Although they share a common catalytic mechanism, each can be distinguished by use of suitable substrates or inhibitors or both (1, 2). The substrates selected here have been used previously to distinguish between certain cysteine-type proteinase activities (2). However, there is some overlap in the specificities for the substrates between the enzymes, and since the assays were performed on crude homogenates rather than on purified proteins, the activities measured in this study will be referred to as, for example, cathepsin B-like. Initial observations from heavily infected animals indicated elevated levels of a range of proteinase activities, most notably, cathepsin H-like activity, the identity of which was supported by the action of the inhibitor E64 [3-L-carboxytrans - 2,3 - epoxypropionylleucyl - amido - (4 - guanidino)butane]. In an attempt to identify whether the source of the proteinases was the host or P. carinii, the activities were followed over the time course of the acquisition of the infection. A close correlation was demonstrated between the cathepsin H-like activity and the level of infection in the lung. A high specific activity was found in lysates of purified P. carinii, and isoelectric focusing (IEF) gels showed bands of cathepsin H-like activity specifically associated with the organism. Furthermore, treatment with co-trimoxazole, which cleared the infection from the lung, caused a marked reduction in cathepsin H-like activity. The cathepsin H-like activity may thus represent a useful marker for diagnosing the presence of P. carinii. The possible significance of these findings is discussed in more detail later.
Corresponding author. 3581
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HAYES ET AL. MATERIALS AND METHODS
Animals. Male Sprague-Dawley rats (Harlan-Olac Ltd., Bicester, Oxon, United Kingdom [U.K.]), with an initial body weight of 220 g, were randomly divided into control and treated groups of three and six animals, respectively. The treated groups were immunosuppressed by treatment with dexamethasone given in their drinking water at a concentration of 2 mg/liter (plus 500 mg of tetracycline per liter as antibiotic protection against other infections [20]). Control and treated animals were housed separately, and groups were sacrificed after 1, 2, 3, 4, 5, 6, 8, 10, and 12 weeks of treatment. In a separate series of experiments, groups of animals on the immunosuppressive regimen were treated with co-trimoxazole (Septrin pediatric suspension; Wellcome Foundation Ltd., Dartford, Kent, U.K.) given in the drinking water at the following dose for 5 weeks following confirmation of a heavy infection: trimethoprim, 50 mg/kg/day; sulfamethoxazole, 250 mg/kg/day. Tissue preparation and identification of P. carinii cysts. The animals were anesthetized with pentobarbital (60 mg/kg, intraperitoneally), the hepatic portal vein was cannulated, and perfusion was commenced with phosphate-buffered saline (PBS) (4°C) while the lungs were perfused with PBS via the pulmonary artery. The lungs were removed from the animal, impression smears were made from each lobe, and the tissue was placed on ice prior to freeze-clamping. The samples were stored at -70°C, and enzyme activities were assayed within 14 days. Cysts of P. carinii were identified in the lung smears by using an indirect, fluorescently labelled antibody kit (Detectif Pneumocystis carinii; Northumbria Biologicals Ltd., Northumberland, U.K.). Each smear (at least 10 fields per lobe) was examined under a fluorescence microscope, and the total number of cysts on the smear was estimated and averaged for each lung. This method of histological scoring of impression smears has been validated elsewhere and shown to correlate closely with total numbers of cysts in lung homogenates (22). The level of infection was scored by using the following criteria: 0, no cysts; 1+, cyst count of 1 to 10, very mild infection; 2+, cyst count of 11 to 1,000, mild infection; 3+, cyst count of 1,001 to 10,000, moderate infection; 4+, cyst count of >10,000, heavy infection. The lung smears and preparations of P. carinii (see below) were free of bacterial and fungal infection at the light-microscope level. Homogenization. Frozen lung samples were thawed on ice and homogenized in 100 mM phosphate buffer containing 2 mM EDTA, pH 6.0, with an Ultraturrax homogenizer (Janke and Kunkel, from BDH Ltd., Poole, Dorset, U.K.) used at maximum speed for two 20-s periods. The homogenate was centrifuged at 2,000 x g for 10 min, and the resulting supernatant was then spun at 14,000 x g for 5 min in a Microfuge. Enzyme activities were determined in the 14,000 X
g
supernatant.
Purification of P. carinii. P. carinii was purified from the lungs of rats maintained on the dexamethasone treatment for >10 weeks by using a unit gravity sedimentation method based on that of Taylor and Easmon (43). Briefly, perfused lungs were finely minced in 100 ml of RPMI 1640 medium containing 40,000 U of penicillin, 40 mg of streptomycin, 6,000 Kunitz units of DNase I from bovine pancreas, and 3,500 U of heparin from bovine lung. The tissue was homogenized in a stomacher (Lab-Blender; Seward Medical, London, U.K.) for 1 min and, after standing on ice for 30 min,
passed through a steel tissue filter (size, 60 mesh; Sigma Chemical Co. Ltd., Dorset, U.K.). Following centrifugation
INFECT. IMMUN.
at 2,000 x g for 10 min (4°C), the pellet was resuspended in about 15 ml of RPMI 1640 containing 0.5% (vol/vol) Histopaque 1077. This was layered onto a 400-ml Histopaque 1077 gradient (1 to 3%, also prepared in RPMI 1640) on a cushion of 0.5 M sucrose (200 ml, in RPMI 1640) in a purposely built Perspex chamber (43) and left to stand for at least 4 h at 4°C. The P. carinii-rich fractions (between 500 and 580 ml as collected from the base of the gradient) contained >1,000 P. carinii per rat cell nucleus, and these were washed in PBS and pelleted by centrifugation at 2,000 x g for 10 min. The combined pellets were resuspended in 100 mM phosphate buffer-2 mM EDTA, pH 6.0, and the cells were lysed by three cycles of freeze-thawing followed by sonication at 50% power for two 20-s periods (Soniprep 150; MSE, Crawley, Sussex, U.K.). The lysate was centrifuged at 14,000 x g for 5 min, and the supernatant was assayed for enzyme activity. Enzyme assays. Cathepsin B, H, and L activities were determined fluorometrically at 37°C as detailed by Barrett and Kirschke (2). Briefly, cathepsin B was measured at pH 6.0 (100 mM phosphate buffer containing 2 mM EDTA and 2 mM dithiothreitol [DTT]) with 20 ,uM N-benzyloxycarbonyl (Z) - Arg - Arg - 7 - amino - 4 - methylcoumarin (AMC) as substrate; cathepsin L was determined at pH 5.5 (100 mM acetate buffer containing 2 mM EDTA and 2 mM DTT) with 20 ,uM Z-Phe-Arg-AMC; and cathepsin H was measured at pH 6.8 (100 mM phosphate buffer containing 2 mM EDTA and 2 mM DTT) with Arg-AMC (20 ,uM). Elastase activity was determined at pH 6.8 (100 mM phosphate buffer containing 2 mM EDTA and 2 mM DTT) with succinyl-Ala-AlaPhe-AMC (20 ,uM) as substrate (5). Lysozyme activity was assessed at 25°C by following the decrease in A450 of a suspension of Micrococcus lysodeikticus in phosphate buffer, pH 6.5 (39). Acid phosphatase was assayed at 37°C by using a Sigma kit (no. 435). Protein concentration was measured by the Bio-Rad method as described by the suppliers (Bio-Rad Laboratories, Watford, Herts, U.K.). IEF and detection of proteinase activity. IEF was performed by using ultrathin gels essentially as described by Radola (33). Ultrathin gels, 5% T and 3% C, were prepared with pH ranges of 4 to 8 (LKB ampholines; LKB Ltd., Milton Keynes, Bucks, U.K.), using a Pharmacia PBE-3000 system (Milton Keynes). After focusing (2 h, 2,000 V), proteinase activity was demonstrated with enzyme overlay strips as detailed by Smith et al. (41). Briefly, cellulose acetate strips were soaked in buffer containing 0.1 mM substrate (as described above) and 2 mM DTT. These were placed over the gels and incubated at 37°C in a humidified chamber. Activity was detected on the acetate strips as fluorescent bands under UV light (366 nm). Materials. RPMI 1640 medium, penicillin, and streptomycin were obtained from GIBCO BRL, Paisley, Renfrewshire, U.K. All other reagents were purchased from Sigma Chemical Co. Ltd. or BDH Ltd. unless stated otherwise in the text. Statistical methods. Enzyme activity is expressed as nanomoles per minute per gram (wet weight), except that for lysozyme, which was standardized by measurement of egg white lysozyme (Sigma L-6876). The activities are given as the mean + standard error of the mean. Statistical significance was calculated by use of Student's t test and reported as significant when the P value was
