Cathepsin B synthesis by the HL60 promyelocytic cell line: effects of stimulating agents and anti-inflammatory compounds

Biochimica et Biophysica Acta 887 (1986) 283-290

283

Elsevier BBA 11635

Cathepsin B synthesis by the HL60 promyelocytic cell line: effects of stimulating agents and anti-inflammatory compounds David Burnett

a,b,

John Crocker c, Simon C. Afford a, Christopher M. Bunce Geoffrey Brown b and Robert A. Stockley a

b,

'~ The Lung Immunobiochemical Research Laboratory, Clinical Teaching Block, The General Hospital, Steelhouse Lane, Birmingham B4 6NH, h Department of Immunology, University of Birmingham and c Department of Histopathology, East Birmingham Hospital, Birmingham (U. K.)

(Received January 9th, 1986)

Key words: Cathepsin B; Cysteine proteinase; Endotoxin; Anti-inflammatory agent; (Promyelocyte)

Cathepsin B synthesis by the human HL60 promyelocyte cell line was investigated by immunohistochemistry and by the assay of the enzyme in cell lysates using a fluorimetric substrate. HL60 cells were shown to produce cathepsin B in response to treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Intracellular levels of cathepsin B and immunohistochemical staining of the enzyme were related to time in culture with increasing concentrations of TPA from 1 n m o l / ! to 8.0 nmol/I. Synthesis of cathepsin B was associated with TPA-induced phagocytic activity of cells in culture, expression of a-naphthyl acetate esterase and reduced cell division. Cathepsin B production was, therefore, related to differentiation of the HL60 promyelocytes into mature macrophage-like cells. Cathepsin B activity in HL60 cell lysates was significantly increased by incubation of the cells with 10 p g / m l endotoxin (iipopolysaccharide) from Escherichia coli, but not carageenan. The production of cathepsin B by TPA-induced HL60 cells was significantly reduced by 0.25 p m o l / I dexamethasone and the non-steroidal anti-inflammatory compound 4-(6-methoxy-2-naphthyl)-butan-2-one but not by indomethacin. The HL60 promyelocytic cell line is a useful model for the study of factors affecting proteinase synthesis by human mononuclear phagocytes. Introduction Cathepsin B is a lysosomal cysteine proteinase that can digest a number of protein substrates including collagen, proteoglycans and insulin [1]. The enzyme has an acid pH optimum of about 6.3 and a molecular mass of 24-28 kDa [1,2], although larger latent precursor forms have been described [3]. Cathepsin B has been located within human macrophages [4,5,6], fibroblasts [6,7], neurones [6], myocytes [8] and epithelial cells of several organs including endocrine glands [6]. The enzyme is probably involved in the intracellular degradation of proteins [9] and it has been proposed to have a role in the processing of hormones within

endocrine tissues [10]. In view of its acid pH optimum and instability at neutral pH [2] it is not known whether cathepsin B functions extracellulary. However, cathepsin B-like activity has been described in epithelial tumours and it has been suggested that the enzyme may have a role in tumour invasion and metastasis [11]. Cathepsin B-like activity has also been detected in lung secretions [4,12], ascitic fluid [3] and synovial fluid [13] and could potentially contribute to extracellular connective tissue damage during inflammation. At present the full biological role of cathepsin B is uncertain but its presence in many tissues suggests it has important functions. The factors controlling the synthesis of cathep-

0167-4889/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

284 sin B and other lysosomal proteinases are not fully understood although they are important to our understanding of many normal and pathological cellular processes. Morland [14] has shown that cathepsin B activity increases when human monocytes mature into macrophages in vitro. Furthermore, the enzyme activity was increased when monocytes were incubated with bacterial endotoxin or carageenan. We have shown previously that the human HL60 promyelocytic cell line can be stimulated, by the phorbol ester 12-O-tetradecanoylphorbol 13acetate (TPA), to synthesise cathepsin B [15]. This cell line may therefore provide a useful model for the study of factors which influence the synthesis of lysosomal enzymes by mononuclear phagocytes. The purpose of the present study was to investigate cathepsin B synthesis during TPA-induced differentiation of HL60 promyelocytes into monocyte-like cells. In particular, we wished to verify the association between enzyme synthesis and differentiation by measuring the known markers of differentiation, namely expression of c~-naphthyl esterase, phagocytic activity and decreased cell division. Furthermore, since bacterial endotoxin and carageenan can stimulate human monocytes to increase cathepsin B production [14], kve investigated the effects of these stimulating agents on HL60 cells for comparison. Corticosteroids have been shown to inhibit the maturation of monocytes into macrophages in vitro [16] and to decrease some lysosomal enzyme activities in these cells [17]. We have, therefore, also studied the effects of dexamethasone and two non-steroidal anti-inflammatory compounds on cathepsin B synthesis by TPA-induced HL60 cells in order to assess further the value of these cells as a model for the synthesis of proteinases by macrophages. Methods and Materials

Effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on HL60 cell differentiation and cathepsin B synthesis Preparation of cell cultures. The HL60 cell line, derived from cells obtained from a patient with human promyelocytic leukaemia, was originally

supplied by Dr. R.C. Gallo, National Cancer Institute, NIH, Bethesda, U.S.A. The HL60 cells were seeded into Linbro cell culture wells (Flow Labs. Ltd.) at a density of 3.5 • 105 cells/ml (2 ml per well) in RPMI 1640 medium (Gibco) supplemented with 10% (v/v) fetal calf serum (Gibco). Cultures were treated with various concentrations (0, 0.5, 1.0, 2.0, 4.0, 8.0 nmol/1) of 12-O-tetradecanoyiphorbol 13-acetate (TPA) (Sigma Chemical Co.) for 16, 40 and 64 h when the cells were harvested [15], and viable cells were counted with a haemocytometer with phase contrast microscopy. Staining for nonspecific esterase activity. Cytocentrifuge preparations of control and TPA-treated cells were stained for a-naphthyl acetate esterase [18]. Percentages of cells staining positively for a-naphthyl acetate esterase were counted. Phagocytic activity. The control HL60 and TPA-treated cells were resuspended at 1 0 6 cells/ml in RPMI 1640 medium containing 10% (v/v) fetal calf serum and 10% (v/v) human AB serum. Gluteraldehyde fixed yeast cells (Saccharornyces cerevisiae) were pre-coated with sheep complement by incubation for 30 min at 37°C with sheep serum. The yeast cells were washed three times and added to the HL60 suspensions at 4 0 . 1 0 6 yeast cells/ml. The H L 6 0 / y e a s t suspension was incubated for 30 min at 37°C and the percentage of cells that had phagocytosed more than three yeast particles was determined by phase-contrast microscopy. Assay of cathepsin B in cell lysates. Each cell preparation (1.5 ml) was centrifuged at 400 × g for 10 min and the cell pellets were washed twice with 0.9% (w/v) NaC1. The washed cells were finally suspended in 1.0 ml of 1 m m o l / l phosphate buffer (pH 6.3) containing 0.2% (v/v) Triton X-100, incubated for 30 min at 40C, frozen to - 7 0 ° C , thawed, and centrifuged at 600 × g for 10 min. The cell lysates were collected and stored at - 70 * C until analysis. Cathepsin B activity in the cell lysates was measuring using a fluorimetric assay with the substrate N-a-CBZ-arginyl-arginine-4-methyl-7-coumarylamide (N-a-CBZ-Arg-Arg-MEC; Bachem, Switzerland). Cell lysates (0.05-0.2 ml) were incubated for 5 h at 37°C in 1.0 ml of 0.1 m o l / l phosphate buffer

285 (pH 6.3) containing 0.2% (v/v) Triton X-100, 1 mmol/l ethylenediamine tetracetic acid, 0.1 mmol/l dithiothreitol and 1.5 /xg of the substrate N-a-CBZ-Arg-Arg-MEC. The reaction was stopped by adding 1 ml 0.1 mol/1 iodoacetic acid in 1 mol/1 Tris-HC1 buffer (pH 8.0). The product of the reaction, 7-amino-4-methylcoumarin was measured with a Perkin-Elmer model 1000 M spectrofluorimeter at an excitation wavelength of 382 nm and emission wavelength of 473 nm. Each sample was simultaneously assayed in the presence of the cysteine proteinase inhibitor N-aCBZ-Phe-Ala-diazomethylketone [19] (Bachem, Switzerland) at a concentration of 2 • 1 0 - 6 mol/1. Enzyme activity was calculated as that inhibited by N-a-CBZ-Phe-Ala-CHN 2 and expressed as fig aminomethylcoumarin generated per h of incubation by 1 ml of lysate obtained from 1 0 6 cells. A calibration line was obtained by using dilutions of the reaction product, aminomethylcoumarin (Protein Research Foundation, Osaka, Japan). Quantitation of cathepsin B-containing cells by immunohistochemistry. Suspensions of control HL60 cells and those incubated with TPA were centrifuged with a Shandon cytocentrifuge. These cytocentrifuge preparations were fixed for 48 h in 10% (v/v) formal-saline containing 10% (v/v) acetic acid. The preparations were washed in 0.1 mol/1 Tris-HCl-saline buffer (pH 7.6). They were then treated with normal swine serum, 20% (v/v) in Tris-HCl-saline buffer (pH 7.6) and then incubated with sheep anti-human cathepsin B [4,5,6], 1% (v/v) in Tris-HCl-saline buffer for 45 min at room temperature. The cell preparations were then washed in Tris-HCl-saline-sucrose (pH 7.6) and incubated with swine anti-sheep IgG, 1% (v/v) (Serotec Ltd., Bicester, Oxon, U.K.) for 45 min, washed and incubated with sheep peroxidase-antiperoxidase complex (Serotec), 2.5% (v/v) for 45 min. After further washing, the preparations were treated for 5 min with 3,3'-diaminobenzidine tetrahydrochloride dissolved (5 mg/10 ml) in Tris-HCl-saline (pH 7.6) to which one drop of 100 vol. (30%) hydrogen peroxide was added. The preparations were counterstained with Mayer's haemalum for 20 s, dehydrated, cleared and mounted in synthetic medium. The cell preparations were examined microscopically at X 40

magnification. At least 200 cells were counted and the percentage of cells stained for cathepsin B was calculated.

Effect of endotoxin, carageenan and anti-inflammatory agents on cathepsin B synthesis Endotoxin and carageenan. Six replicate experiments were performed. The HL60 cells were seeded at 3.5 • 105 cells/ml into Linbro wells as described above. As well as control cultures, the cells were also incubated with: (a) 10 ~g/ml endotoxin (lipopolysaccharide) from E. coli (strain 026:B6) obtained from Sigma Chemical Co, Poole, U.K., and (b) 25 #g/ml carageenan type IV lambda from Gigartina aciculaire and G. pistillata (Sigma Chemical Co.) After 48 h of incubation, the cells were harvested, counted and lysates were prepared and assayed for cathepsin B as described above. Differences between cathepsin B activity in lysates from control cultures and those incubated with endotoxin or carageenan were tested using Wilcoxon's test for paired data. Effects of anti-inflammatory agents on cathepsin B synthesis by TPA-induced cells. Six replicate experiments were performed. HL60 cells were cultured for 48 h in the presence of 8.0 nmol/1 TPA (controls) and in the presence of: (a) 0.25/~mol/1 dexamethasone (Sigma Chemical Co.); (b) 0.25 /~mol/1 indomethacin (Sigma Chemical Co.), and (c) 0.25 #mol/1 of the non-steroidal anti-inflammatory agent 4-(6-methoxy-2-naphthyl)butan-2-one (nabumetone) supplied by Beechams Research Laboratories, U.K. The cell lysates were prepared and analysed for cathepsin B activity as described above. Results

Effect of TPA on HL60 differentiation and cathepsin B synthesis Morphology and cell division. Cytocentrifuge preparations of untreated HL60 cells showed the typical morphological features associated with promyelocytes. As described previously [15], the cells had granular cytoplasm and large, sometimes double nucleolated nuclei. HL60 cultures treated for 64 h with TPA contained a reduced number of cells as compared with control cultures (Fig. 1).

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Fig. 1. HL60 cell numbers (per ml) after 0, 16, 40 and 64 h in culture in the absence of TPA (O) or in the presence of TPA at 0.5 n m o l / l (©), 1.0 nmol/1 (A); 2.0 nmol/1 (n); 4.0 nmol/1 (,~) and 8 nmol/1 (U). Cells were originally seeded at 3.5-103 cells/ml.

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Fig. 3. The percentage of HL60 cells which phagocytosed opsonized yeast, after culture for up to 64 h in medium without TPA (O) or in the presence of TPA at 0.5 n m o l / l (©), 1.0 n m o l / l (-); 2.0 nmol/l (n); 4.0 nmol/I (,~) and 8 nmol/I (m).

The reduced growth of TPA treated cells was dose dependent and consistent with maturation o f the cells into mature non-dividing monocyte-like cells [20,21]. These mature cells had rounded reniform nucleolated or non-nucleolated nuclei, often compressed by one or more large cytoplasmic vacuoles. a-Naphthyl acetate esterase staining. Fig. 2 shows

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Time in culture (hr) Fig. 2. Percentage of HL60 cells staining for a-naphthylacetate esterase activity after culture for up to 64 h in medium without TPA (O) or in the presence of TPA at 0.5 n m o l / l (©); 1.0 n m o l / l (A); 2,0 nmol/l (D); 4.0 nmol/1 (~) and 8 nmol/1 (m).

Fig. 4. Cathepsin B activity in lysates prepared from HL60 cells after culture for up to 64 h in control medium (O) or in the presence of TPA at 0.5 nmol/1 (©); 1.0 nmol/l (A); 2.0 n m o l / l (0); 4 nmol/1 (~); 8 nmol/l (m). Enzyme activity (ordinate) is expressed as ng 7-aminomethylcoumarin (AMC) in 1 ml of lysate from 1-106 cells after 1 h of incubation with substrate.

287 the percentage of cells stained for a-naphthyl acetate esterase. Control cells demonstrated no staining for a-naphthyl acetate esterase. Addition of increasing amounts of TPA to cultures induced a dose-dependent expression of a-naphthyl acetate esterase activity. A maximum of 75-80% positive cells was observed 64 h after incubating cells with 2.0-8.0 nmol/1 TPA. Phagocytosis of opsonized yeast. In control HL60 cultures less than 1% of cells were observed to phagocytose yeast particles. Cultures treated with TPA contained increased numbers of phagocytic cells which was both dose and time dependent; 33-35% of the cells were phagocytic after 64 h incubation with TPA at 2.0-8.0 nmol/1 TPA (Fig. 3). Cathepsin B activity in cell lysates. Cathepsin B activity was absent in control HL60 cells, although low levels were observed in lysates after 64 h in culture. In contrast, significant cathepsin B activity was observed in cells treated with TPA. The induction of cathepsin B activity was dose and time dependent at TPA concentrations in excess of 1.0 nmol/1 (Fig. 4).

Irnmunohistochemical staining of cells for cathepsin B. Fig. 5 shows the results of immuno-

histochemical staining of the HL60 cells for cathepsin B, before and after 64 h culture with TPA. Fig. 6 shows the proportion of cells demonstrating cathepsin B as revealed by immunoperoxidase staining with a monospecific antiserum to the enzyme. Only 3% of the control cells stained positively for cathepsin B after 64 h in culture. However, in parallel with cathepsin B activity in cell lysates, treatment of cells with TPA at concentrations in excess of 1 nmol/1 was associated with a dose and time dependent increase in the number of cells expressing cathepsin B antigen. A maximum of 79% positive cells was observed 64 h after culture with 8 nmol/1 TPA.

Effects of bacterial endotoxin, carageenan and antiinflammatory agents on cathepsin B synthesis Endotoxin and carageenan. Table I shows the average cathepsin B activity in cell lysates from control unstimulated HE60 cells and those incubated with 10 /~g/ml endotoxin or 25 ~ g / m l carageenan. Enzyme levels were increased in all six cultures treated with endotoxin ( P < 0.01) but in only three of those incubated with carageenan ( P = n.s.).

(b) Fig. 5. Immunohistochemicalstaining of HL60 cells with sheep anti-human cathepsin B. (a) Control HL60 cells which were negative for cathepsin B, (b) HL60 cells incubated for 64 h in medium containing 8 nmol/l TPA. Most of the TPA-induced cells demonstrate intra-cellular staining for cathepsin B although a few negative cells are shown (n).

288

nabumetone are shown in Table I. All six cultures incubated with TPA + 0.25 ~ m o l / l dexamethasone had decreased enzyme levels compared to the cultures treated with TPA alone (mean decrease 44%, S.D. 32.8%; P < 0.01). Incubation of cell cultures with TPA + 0.25/~mol/1 nabumetone was associated with decreased intracellular cathepsin B activity in five of the six experiments (mean decrease 22.1%, S.D. 36.3%; P < 0.05). In addition, a decrease in cathepsin B was also observed in five of the six cultures incubated with TPA + 0.25 /~mol/1 indomethacin (mean decrease 21.1%, S.D. 29.5%) but this did not achieve statistical significance.

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Discussion Fig. 6. The proportion of cells (%) stained for cathepsin B after culture for up to 64 h in control medium (o), or medium containing TPA at 0.5 n m o l / l (O); 1.0 nmol/1 (A); 2 n m o l / l (t2); 4 nmol/1 (~) or 8 n m o l / l (11).

Dexamethasone, indomethacin and nabumetone. The average cathepsin B levels in cell lysates from control, TPA-treated HL60 cells and the cultures incubated with dexamethasone, indomethacin and TABLE I EFFECTS OF S T I M U L A T I N G A G E N T S A N D ANTI-INF L A M M A T O R Y C O M P O U N D S ON C A T H E P S I N B ACTIVITY IN HL60 C E L L LYSATES All cells were cultured for 48 h without TPA (unstimulated), or with 8.0 n m o l / T P A , n.s., not significant. Cell cultures

Unstimulated control HL60 Unstimulated + 1 0 / ~ g / m l endotoxin Unstimulated + 2 5 / x g / m l carageenan T P A 8 nmol/1 only TPA + 0.25/L m o l / l dexamethasone T P A + 0.25 # mol/1 nabumatone TPA + 0.25 # mol/1 indomethacin

Mean cathepsin B activity + S.D. (ng aminomethylcoumarin/106 cells/ml per h)

Statistical significance

14.7 + 11.6 34.5 + 7.7

P < 0.01

28.9 + 24.1

n.s.

295.4+ 196.4 155.0 5:104.4

P < 0.01

172.6 + 68.9

P < 0.05

198.6 _+62.6

n.s.

The HL60 cell line was derived from the peripheral blood leucocytes of a patient with acute promyelocytic leukaemia [20]. Most of the cells are promyelocytic although about 10% were reported to have the morphological features of mature myeloid cells [21]. The addition of polar compounds such as dimethylsulphoxide to HL60 cells induced differentiation towards neutrophilis [21]. However, these neutrophils are deficient in secondary granules and their associated proteins [22]. In contrast, the HL60 cells can be induced to differentiate into mature monocyte-like cells by phorbol esters [23] or 1,25-dihydroxyvitamin D-3 [24]. This differentiation is associated with increased cell adherence, phagocytic activity, lgG receptor expression and the synthesis of enzymes which are found in mature monocytes, including acid phosphatase [23,25], NADase [21] and thromboplastin-like procoagulant activity [26]. The HL60 cell line therefore represents a useful model for the study of enzyme synthesis by human macrophages. Previously, we have shown that phorbol ester (TPA) treatment of HL60 cells resulted in cathepsin B synthesis by these cells [15]. The present study investigated the relationship between TPAinduced cathepsin B synthesis and other parameters of HL60 differentiation towards macrophages. Our previous study [15] employed a cathepsin B assay utilising the fluorimetric substrate benzoyloxycarbonyl-L-phenylalanyl-L-arginine-4-methyl7 - c o u m a r y l a m i d e ( N - a - C B Z - P h e - A r g - M EC)

289 which was originally described as a specific cathepsin B substrate [27]. However, N-a-CBZArg-Arg-MEC has since been shown to be a better substrate for cathepsin B [1] and it was therefore substituted for the present study. The specificity of the assay for cysteine proteinase activity was further enhanced by measuring the enzyme activity inhibited by the potent inhibitor, N-a-CBZPhe-Ala-diazomethylketone [19]. This procedure was used to exclude any nonspecific hydrolysis of the substrate by other classes of proteinase [27]. The results of the study using enzymatic and immunohistochemical methods, revealed that HL60 cells, when incubated with TPA, show increased expression of cathepsin B. The induction of cathepsin B synthesis by the cells was related to the amount of TPA added and time in culture. Enzyme activity was associated with increased phagocytic activity of cells in culture and expression of a-naphthyl acetate esterase activity, confirming that cathepsin B synthesis was intimately linked to differentiation of the HL60 promyelocytes into mature non-dividing monocyte-like cells. The results are relevant to normal non-malignant mononuclear phagocytes since M~rland has shown that cathepsin B synthesis increases in peripheral blood monocytes as they mature into macrophages during culture [14]. Furthermore, bacterial endotoxin was shown to increase the cathepsin B activity in monocytes although the effects varied in cultures from different donors [14]. The present study shows that cathepsin B synthesis by HL60 cells is sensitive to stimulation with endotoxin from E. coli. However, carageenan, which stimulates cathepsin B activity in monocytes [14], had no significant effect on enzyme levels in HL60 cells. Dexamethasone has an inhibitory effect on the maturation of monocytes into macrophages [17] and this is associated with some decreased intracellular enzyme activities [16]. Incubation of TPA-stimulated HL60 cells with dexamethasone resulted in decreased intracellular cathepsin B activity. It remains to be fully established whether the corticosteroid reduced the response of HL60 cells to differentiation by TPA, or if it caused a reduction in lysosomal enzyme synthesis despite cell differentiation. Cell counts in TPA-induced

cultures at 48 h (mean 3.8.105 cells/ml, S.D. 0.10) were similar to those of cultures treated with dexamethasone together with TPA (mean 3.5 • 105 cells/ml; S.D. 0.11). These observations would therefore suggest that dexamethasone did not inhibit TPA-induced differentiation, since this would have been associated with continued cell division. Dexamethasone may, therefore, influence the degree of terminal differentiation of HL60 cells cultured with TPA. Alternatively, the corticosteroid may have a more direct effect on the synthesis of some proteins, including cathepsin B, by induced HL60 cells. The non-steroidal compound 4-(6-methoxy-2naphthyl)-butan-2-one (nabumetone) has been shown to inhibit the accumulation of neutrophils and macrophages in experimental rat granulomas and to reduce lysosomal enzyme levels in exudate fluid [29]. When nabumetone was added together with TPA to HL60 cultures a significant reduction in cathepsin B activity was observed in cells at 48 h, demonstrating the potential sensitivity of this cell line to some non-steroidal anti-inflammatory agents. The effects of indomethacin failed to reach statistical significance even though cathepsin B levels were shown to be reduced in five of the six experiments. This may have been due to the variability of the experimental procedures, although previously indomethacin has been shown not to inhibit TPA-induced HL60 differentiation [29]. In conclusion, HL60 promyelocytic cells cultured in the presence of TPA showed increased cathepsin B activity which was associated with morphological and functional differentiation towards more mature monocyte-like cells. The influence of some stimulating and anti-inflammatory agents on cathepsin B synthesis by HL60 cells was largely similar to that observed in studies of human monocytes, but the mechanisms of their actions require further study. The HL60 cell line represents an appropriate model for the study of factors which influence lysosomal enzyme synthesis by human mononuclear phagocytes.

Acknowledgements The authors would like to acknowledge the support of the Birmingham Hospitals Endowment Fund, The Medical Research Council (U.K.),

290 Bencard

and

t h e E.E.

Lawley

Foundation.

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t h a n k M r s . J. S h a w f o r t e c h n i c a l a s s i s t a n c e a n d M i s s K. R o b e r t s f o r t y p i n g t h e m a n u s c r i p t .

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