1073

result in clinically demonstrable infection. Elek4 has shown that subcutaneous injection of 105 Staphylococcus aureus bacteria did not lead to pus formation in healthy volunteers. A minimum dose of 7-3x10 cocci was needed for pus formation to occur after intradermal injection.4 In our series the average number of bacteria 3 on an uncleansed skin site was 1-3 x 103 bacteria/5-3 cmzarea; the highest individual value was 2 x 104 bacteria/5-3 cm2. When we take into account the tiny surface area needed for insertion of the small needle used in insulin injections, it is apparent that the number of bacteria injected in an uncleansed site is well below that needed for pus formation. The minimum doses of bacteria required to induce pus formation in normal subjects may not apply to diabetics, who are more susceptible to infection.5 Furthermore, the relatively small number of subjects in our study precludes our recommending the routine elimination of skin preparation before insulin injection. However, this study does indicate that skin preparation with alcohol is not always necessary, particularly in individuals with good personal hygiene. Omitting skin preparation when it is not necessary saves time and money and

Preliminary Communication ACID HYDROLASES IN MONOCYTES FROM PATIENTS WITH INFLAMMATORY BOWEL DISEASE, CHRONIC LIVER DISEASE, AND RHEUMATOID ARTHRITIS N. K. GANGULY J. G. C. KINGHAM B. LLOYD R. S. LLOYD C. P. PRICE D. R. TRIGER* RALPH WRIGHT

Southampton University Hospitals A sensitive

technique was used to estiacid hydrolases—N-acetyl-&bgr;glucosaminidase (N.A.G.) and &bgr;-glucuronidase (B.G.) produced by peripheral-blood monocytes. Enzyme levels were measured after in-vitro incubation of monocytes with or without stimulation by zymosan and endotoxin. Compared with controls, enzyme production and release in inflammatory bowel disease, chronic liver disease, and rheumatoid arthritis were markedly raised. It is suggested that various stimuli, including immunological ones, may be responsible for the release of such. enzymes from monocytes and that such release may be a factor in the production of the chronic inflammation seen in these disorders. Summary

mate two

INTRODUCTION

CHRONIC inflammation is responsible for the tissue damage in many diseases, such as int1ammatory bowel disease, chronic liver disease, and connective-tissue diseases. Mononuclear phagocytes are present at the site of tissue damage and are thought to be involved in the pathogenesis and resolution of the disease process.’ Acid

hydrolases produced by macrophages can cause extensive tissue damage when released into their immediate

pain associated with the injection of unevaporated alcohol into the skin. Many diabetics have to take their afternoon or evening insulin dose when they are out of their homes. Avoidance of skin preparation may make this injection easier, thus increasing motivation to change from a single dose to two which in some cases improves metabolic daily injections, 6 helps to

avoid

some

of the

control.

We thank Prof. Richard Root, for advice and laboratory facilities for the bacterial cultures. V.A.K. is recipient of a grant from the Finnish Ministry of Education and P.F. is an established investigator of the American Diabetes Association. This study was supported in part by grants AM13526 and RR125 from the National Institutes of Health.

Requests for reprints should be addressed to P.F., Yale University School of Medicine, Department of Internal Medicine, 333 Cedar St., New Haven, Connecticut, 06510, U.S.A. REFERENCES T. C.

1. 2. 3.

Dann, Lancet, 1969, ii, 96. Williamson, P., Kligman, A. M. J. Invest. Dermat. 1965, 45, 498. Snedecor, G. S., Cochran, W. G. Statistical Methods. p. 130. Ames, Iowa,

4. 5. 6.

Elek, S. O. Ann. N. Y. Acad Sci. 1956, 65, 85 Robertson, H. D., Polk, H. C. Surgery, 1974, 75, 123. Didier, J., Eschwege, E., Guyot-Argenton, C., Aubry, J.-P., Tchobroutsky, G. Diabetes, 1976, 25, 463.

1967.

environment.2 The release of hydrolases

can

be achieved

by various immunological means, such as stimulation by immune complexes,3 complement components, or zymosan,4,5by lymphokines released during a delayed hypersensitivity response,6 and by bacterial products.7 Most of these immunological mechanisms operate in the chronic inflammatory conditions mentioned above.8-11However, to the best of our knowledge, there are no reports of the production of acid hydrolases by peripheral-blood monocytes in chronic inflammatory conditions. We therefore studied the production of two acid hydrolases, N-acetyl-p-glucosaminidase (N.A.G.) and j3-glucuronidase (B.G.), by purified peripheral-blood monocytes in patients with inflammatory bowel disease (I.B.D.), chronic liver disease (L.D.), rheumatoid arthritis (R.A.) and systemic lupus erythematosus (S.L.E.), and in normal controls. Production of these enzymes was measured before and after stimulation with endotoxin and zymosan. PATIENTS AND METHODS

Patients bowel disease.-12

Inflammatory studied; 7 hadulcerative receiving

patients with

I.B.D. were

All studied; 7 had ulcerative colitis and 35 had Crohn’s disease. AH

corticosteroids at the time of study. Liver disease.-This group comprised 8 cases of alcoholic cirrhosis, 3 cases of chronic active hepatitis, and 1 case of alcoholic hepatitis, diagnosed on standard clinical and histological criteria. RA. and S.L.E.-There were 10 patients with classic R.A. and 2 patients with s.L.E. as judged by the American Rheumatology Association classification. All the patients were receiving some form of medication; the major drugs being indomethwere

acin, corticosteroids, aspirin, penicillamine, gold, aloxiprin, chloroquine, azathioprine, benorylate, and ibuprofen. Controls.-27 age and sex matched controls were studied. There were 17 members of the laboratory staff and 10 patients attending a day surgical unit for minor surgical procedures. None of the controls had a history or clinical evidence of any inflammatory or hepatic disorder, or had recently been immunised.

Monocyte Isolation * Present address: Department of Medicine, Royal Hospital, Sheffield.

Patients and controls fasted before withdrawal of 20 ml of

1074 blood into siliconised glass bottles containing heparm. purified monocyte suspension was obtained from the heparinised blood by the method of Lloyd and Levick.12 Studies on the suspension showed that more than 95% of the monocytes excluded trypan blue and more than 99% of the cells phagocyvenous

A

tosed ’Fluolite’ DS5005. This indicated the viability and activity of the purified cells. Preparation of Endotoxin and Zymosan Suspensions Stock endotoxin (Salmonella typhosa 0901 lipopolysaccharide, Difco Laboratories) and zymosan (Sigma) preparations in

0-85% sodium chloride were stored at -20°C. at a concentration of 1 mg/ml. For use, the stock preparations were diluted with R.P.M.I. 1640 medium (Gibco Bio-cult, Paisley, Scotland), buffered with 20 mmol/1 N-2-hydroxyethylpiperazine-Nethane sulphuric acid (HEPES), to give a working concentration of 50 p.g/ml. Activation of Monocytes 20 mmol/1 HEPES buffered R.P.M.I. 1640, supplemented with 10% fetal calf serum (S-R.P.M.I. 1640) was used for cell maintenance. 1.5x106 purified monocytes in S-R.P.M.I. 1640 were added to each of the six wells of a sterile plastic multiplate (Lux Scientific Corporation) and allowed to adhere to the surface by incubation at 37 °C. in a 5% carbon dioxide fully humidified atmosphere for 2 h with occasional agitation. An extra well containing a glass coverslip was used for each sample in order to check both cell viability by uptake of fluolite on exclusion of trypan blue and the degree of stimulation and adherence at the end of the stimulation period. After incubation, the supernatant was discarded and two of the wells were filled with 1.55 ml of the working endotoxin solution, two with zymosan suspension, and the remaining two with S-R.P.M.I. 1640 alone. The plates were incubated for a further 2 h and the supernatants discarded. All wells were washed three times with Hanks’ solution (buffered with 20 mmol/1 HEPES) to remove the zymosan and endotoxin, and refilled with 1.5 5 ml of S-R.P.M.I. 1640. The plates were then incubated under the same conditions for 24 h, the supernatants were collected from each well, centrifuged to remove any contaminating cells, and stored in plastic tubes at -20°C. The adherent cells were removed from the plastic surface of each well with a rubber "policeman" into 1.5 ml of sterile, ice-cold, distilled water, and the cell suspension was rapidly frozen and thawed eight times. The distilled water containing the lysed monocytes was examined under a microscope to ensure complete disintegration of the cells, centrifuged, and the supernatant stored at —20°C. It was not practical to carry out a cell-count before TABLE I-MEAN

&bgr;-GLUCURONIDASE

Concentrations of p-glucuronidase and N-acetyl glucosaminidase in cells and supernatants after stimulation with zymosan or endotoxin and without stimulation. Bar represents ±

one

standard deviation.

and we have assumed that the cell number after incubation bore a constant relation to the starting inoculum im each case. The total protein concentration of cell lysate was estimated by the method of Lowry et al.11 and was used as an index of cell massl4 as there were insufficient cells for D.N.A. measurement by standard techniques.

lysis,

Enzyme Estimations A sensitive assay for N.A.G. and B.G., suitable for the estimation of the enzymes in the cell lysate and supernatant obtained from 1.5 x 106 monocytes, was developed from the spectrofluorometric method of Beutler et Al., 14 with the following modifications: the incubation mixtures contained 1 ml of a 3 mmol/1 solution of 4-methylumbelliferyl-p-D-glucopyranoside LEVELS IN CELLS AND SUPERNATANTS

TABLE II-MEAN N-ACETYL-GLUCOSAMINIDASE CONCENTRATIONS IN CELLS AND SUPERNATANTS

Comparison with controls:* P