LYMPHOCYTE ABNORMALITY IN CHRONIC MUCOCUTANEOUS CANDIDIASIS

1259 their patients; and to Dr. A. Kopec for details of the ABO bloodgroups of the control population. Requests for reprints should be addressed to M. J. S. L.,

University Department of Medicine, General Hospital, Nottingham NG16HA. REFERENCES 1.

2. 3. 4.

5. 6. 7.

8. 9.

Jick, H., Shone, D., Westerholm, B., Inman, W. H. W., Vessey, M. P., Shapiro, S., Lewis, G. P., Worcester, J. Lancet, 1969, i, 539. Kopec, A. Personal Communication. Dick, W., Schneider, W., Brockmuller, K., Mayer, W. Thromb. Diath. hœmorrh. 1963, 9, 472. Lancet, 1966, ii. Aird, I., Bentall, H. H., Roberts, J. A. F. Br. med. J. 1953, i, 799. Roberts, J. A. F. Brit. J. prev. soc. Med. 1957, 11, 107. Clarke, C. A., Cowan, W. K., Edwards, J. W., Howel Evans, A. W., McConnell, R. B., Woodrow, J. C., Sheppard, P. M. Br. med. J. 1955, ii, 643. Langman, M. J. S., Doll, R. Gut. 1965, 6, 270. Merikas, G., Christakopoulos, P., Petropoulos, E. Am. J. dig. Dis. 1966, 11, 790.

10. Horwich, L., Evans, D. A. P.- McConnell, R. B., Donohoe, W. T. Gut, 1966, 7, 680. 11. Evans, D. A. P., Horwich, L., McConnell, R. B., Bullen, M. F. ibid. 1968, 9, 319. 12. Bronte-Stewart, B., Botha, M. C., Krut, L. H. Br. med. J. 1962, i, 1646. 13. Allen, T. M., Dawson, A. A. Br. Heart J. 1968, 30, 377. 14. Preston, A. E., Barr, A. Br. J. Hœmat. 1964, 10, 238. 15. Oliver, M. F., Geizerova, H., Gumming, R. A., Heady, J. A. Lancet, 1969, ii, 605. 16. Langman, M. J. S., Elwood, P. C., Foote, J., Ryrie, D. R. ibid. p. 607.

LYMPHOCYTE ABNORMALITY IN CHRONIC MUCOCUTANEOUS CANDIDIASIS LENNOX HOLT HELGI VALDIMARSSON HARRY R. C. RICHES JOHN R. HOBBS Royal Postgraduate Medical School, London W.12, and Harefield Hospital, Middlesex

Summary

In

a

25-year-old

woman

with chronic

mucocutaneous candidiasis no cutaneous hypersensitivity to candida antigens, tuber-

delayed culin, or dinitrochlorobenzene was found. In vitro challenge of her lymphocytes showed a normal blastoid response to candida, purified protein derivative, and allogenic lymphocytes. This did not, however, result in production of detectable macrophage migrationinhibition factor (M.I.F.). It is suggested that defective production of M.I.F. may be responsible for the clinical manifestations of the disease; and transfusion of competent, histocompatible lymphocytes is proposed. Introduction

IN-vITRO lymphocyte transformation to antigens has been thought to correspond to the delayed-hypersensitivity reaction in vivo.1 Activated lymphocytes produce soluble agents, which can be detected by their mitogenic action on other lymphocytes (M.F.), inhibition of macrophage migration (M.I.F.), cytopathic effect (L.T.), and erythrogenic action on injection into skin. The generic term lymphokines has been given to those substances.2 They are thought to be involved in the

production of delayed-hypersensitivity reactions. In chronic mucocutaneous candidiasis, which has recently received considerable attention.3-6 there

exists the immunological paradox of normal lymphocyte transformation and defective cutaneous cellular immune responses. Chilgren et awl. have postulated

that failure to produce macrophage migration inhibitory factor (M.I.F.) could account for these findings. However, these workers did not provide experimental evidence to substantiate this interesting hypothesis. In a patient with chronic mucocutaneous candidiasis we have demonstrated defective M.I.F. production.

Case-report The patient, a woman of 25, had had repeated fungal and bacterial infections all her life. At the age of 3 years she had a mastoid operation which healed very slowly, and there had since been frequent subcutaneous abcesses. At 9 years she developed pneumonia complicated by a lung abscess in the right upper lobe. Tubercle bacilli were isolated from her sputum and antituberculous treatment was started. Four years later, a lung cyst was excised from the right upper lobe and was found to contain tubercle bacilli, and a further two years’ chemotherapy was given. When the patient was aged 20, an abscess developed in the right lung; this was excised and was found to be a mycetoma. She was left with a severely damaged right lung and a chronic empyema, which had been continually draining through a pleural fistula for five years. There was no history suggesting a familial immune deficiency. On examination the patient was slightly cyanotic. There was a scaling dermatitis of the face, especially along the hairline, around the nose, and on the ears. She had angulostomatitis and severe glossitis suggestive of a chronic monilia infection. Many nails were deformed, showing hypertrophic changes and dark coloration. Investigations revealed a profuse growth of monilia from the mouth, vagina, skin, and nails. Direct microscopy showed yeast forms only. There was a persistent lymphopenia (500-1000 cells per c.mm.). Other routine investigations were normal except for a slightly raised alkaline phosphatase (20 King-Armstrong units). No evidence of endocrine disorders was found and no autoantibodies were detected in serum.

Materials and Methods Wellcome TC 199 was used in all tissueBurroughs culture work. Phytohæmagglutinin (P.H.A.) batch BR6 was obtained from Burroughs Wellcome. Candida antigens (C.A.) were kindly provided by Dr. J. Faux (Institute of Diseases of the Chest, London), and by Dr. I. G. Murray (London School of Hygiene and Tropical Medicine). Purified protein derivate (P.P.D.) was supplied by the Weybridge Laboratories. 3H-thymidine (specific activity 5Ci per millimole) was obtained from the Radiochemical Centre, Amersham. Serum and salivary immunoglobulins were measured by modified Mancini radial immunodiffusion. Parotid saliva was centrifuged, filtered through a 0.45 µ ’Millipore’ membrane, and concentrated down to 1/5 of the original volume before estimating the immunoglobulins. Phagocytic activity was tested with a qualitative nitrobluetetrazolium (N.B.T.) test,8 Leucocytes were stained for myeloperoxidase.9e Dinitrochlorobenzene (D.N.C.B.) sensitisation was

attempted.lo In-vitro Lymphocyte Transformation The method was essentially the same as that described by Holt et al.11 The lymphocytes were separated from defibrinated blood by aggregation of red blood-cells with 3% gelatin, yielding a suspension of cells 85-95% of which were small lymphocytes, whose viability was 95-98%. Triplicate cultures, consisting of 106 cells suspended in 1 ml. TC 199 enriched with 20% fetal calf serum or autologous serum, were set up. Dose-response curves for both P.H.A. and the candida antigens, using the patient’s lymphocytes, were determined, and the optimal concentra-

1260 tion found was used in subsequent experiments. The cultures were incubated in 5% CO2 and 95% air, P.H.A. stimulated cells being harvested after 72 hours and candida activated cells after 120 hours, all cultures being labelled with 1 µCi 3H-thymidine 24 hours before harvesting. Oneway mixed lymphocyte reaction (M.L.R.) was done,12 the

allogenic lymphocytes being inactivated with’MitomycinC ’ and the cultures harvested after 144 hours. M.LF. Assay A modification of the techniques first described by Thor et al.I3 was used. The glassware was used unsiliconised

throughout. The M.I.F. assay chambers were made by cutting round holes of 9 mm. diameter into squares of silicone rubber sheeting (London Splint Co.). Three of these squares were fixed by sterile silicone grease to an ordinary microscope slide. Having been filled, the chambers were closed by mounting another microscope slide on the top, silicone grease again being used as a fixative. The indicator cells were macrophages from unsensitised guineapigs (pathogenfree). The human M.I.F. inhibits the spontaneous migration of these cells. The guineapig macrophages were obtained 48-72 hours after intraperitoneal injection of sterile liquid paraffin by washing out the peritoneum with TC 199 containing preservative-free heparin (10 units per ml.). The cells were washed three times in TC 199 and finally resuspended in TC 199 containing 15% heat-inactivated fetal calf serum, the concentration being adjusted to approximately 30 x 106 cells per ml. Capillary tubes were filled with this suspension; one end was sealed by heating; and the tubes centrifuged at 900 r.p.m. for 5 minutes and cut at the cell-fluid interface. The end containing the cells was then transferred into the migration chamber, and fixed in position by sterile silicone grease. Human M.I.F. was derived from lymphocytes cultured in medium, 6 x 106 cells per ml., and activated with candida antigens (100 µg. per ml.). The medium was enriched with 15% fetal calf serum. The supernatant was harvested after 48 hours, centrifuged at 3000 r.p.m. for 10 minutes, dialysed, and concentrated 5 times through a pressure gradient in TC 199. Finally the pH was adjusted to 7-2 and the supernatant was sterilised by filtration through 0-22 µ Millipore membrane. TABLE I-IMMUNOGLOBULINS IN SERUM AND SALIVA

(Mg./100 ml.)

Fig. 1—Transformation of the patient’s lymphocytes expressed percentage of the controls. as a

mean

transformation of two normal

The responses were measured tritiated thymidine.

by counting the uptake of

Fig. 2-Migration chambers : A, containing supernatant from activated normal candida-sensitive lymphocytes ; and B, containing supernatant from activated patient’s lymphocytes.

in the saliva. In the serum IgG and IgM were normal, but IgA was near the lower limit of normal (see table I). No precipitating antibodies against candida could be detected in serum. The patient’s polymorphs showed normal myeloperoxidase staining and the N.B.T. test revealed an effective phagocytic response in vitro. There was no delayed hypersensitivity following an intradermal challenge with P.P.D. 1/100 (2 µg.) and candida antigens (100 (J.g.). Six normal subjects regularly gave positive reactions to these antigens. There was slight erythema after a D.N.C.B. test (100 jjLg. per ml.) but no induration-i.e.,

delayed hypersensitivity. lymphocyte transformation experiments, the circulating lymphocytes showed a blastic response within the normal range to P.H.A., candida antigens, P.P.D., and foreign lymphocytes (fig. 1). However,

no

In the

The sterile fluid was either kept at -20°C or used immediately for migration experiments. These were done in triplicate. Four sets of triplicates were used differing only in the composition of the fluid in the migration chambers: Medium with 75% fetal calf serum and 500 µg. candida per ml. II: Supernatant from unstimulated normal human lymphocytes cultured for 48 hours. When this was used as migration fluid, 500 µg. of candida antigens was added per ml. III: Supernatant from normal human candida-sensitive lymphocytes activated with the candida antigens. iv: Supernatant from the patient’s lymphocytes activated with the candida antigens. System I was used as the 100% reference standard. The area of migration was measured at 24 hours and expressed I:

antigen

as a

percentage of the reference migration

area.

Results

Immunoglobulins

were

present in normal

amounts

these cells did not release a detectable amount of M.I.F. into the supernatant after activation with the candida

antigens (fig. 2, table II). Lymphocytes from five normal candida-sensitive individuals have on repeated testing caused 50-80% inhibition in

our

system. TABLE II—M.I.F. ASSAY

1261 Discussion

tried circulating human leucocytes as Initially This did not give reproducible cells.14 indicator results. It has been shown that circulating human leucocytes do not carry out active migration in this system even after a few days in culture." The " migrating " cells in the peripheral blood are mainly lymphocytes 14 which can easily glide on a glass surface because they do not stick to it. Furthermore, migration from splenic explants of sensitised lymphocytes as well as polymorphs is not significantly inhibited by antigens.15 We have, therefore, come to the conclusion that the migration observed in this system is largely based on gravity rather than active cell movements, and the occasional inhibition observed is due to non-immunological factors. We then modified the technique described by Thor et al.13 to detect human M.I.F. in vitro. This is a relatively simple and reproducible assay system. The indicator cells are guineapig macrophages from sterile peritoneal exudates. Human M.I.F., crossing the species barrier between man and guineapig, inhibits the spontaneous migration of these cells. The heterologous design of the assay is necessary because obtaining a good migrating cell population from humans is very difficult.16 It is also usual to have contaminant lymphocytes. Since most humans react to candida antigens, such contaminant cells could result in the local release of M.I.F. within the indicator system itself. Lymphokines can be released from such contaminant lymphocytes within 6 hours of exposure to candida antigen from the test supernatant. 17 The use of pathogen-free guineapigs yields a migrating population of macrophages similarly contaminateq with guineapig lymphocytes, but these are unresponsive to candida antigen, and indeed subsequent skin-testing of the donor we

guineapigs was negative. Microorganisms of the Monilia genus are commonly found on the human body; but the host is normally able to resist invasion by these potentially pathogenic fungi, although the balance is dehcate.18 A relatively mild disturbance in the host’s defence mechanism can easily change it in favour of the commensal, which enters into a parasitic relationship with the host.19 From 80 to 94% of normal adults show delayed hypersensitivity after intradermal challenge with candida antigens,20 indicating the existence of acquired cellular resistance to the candida organisms. The normal blastoid transformation of the lymphocytes in our patient points to the existence of sensitive cells with an intact ability to recognise candida antigens. Their activation by these antigens does not,

however,

result in the production of detectable M.I.F. or not the deficient expression of delayed hypersensitivity in this patient can be related to this failure of M.I.F. production remains to be seen. The significance of the various lymphokines in the eradication of invading organisms is not clear. Our results suggest that chronic mucocutaneous candidiasis, as well as susceptibility to some other infections, can be associated with a failure to produce M.I.F. The infectivity of candida, and most other fungi, is thought to be suppressed predominantly by cellular immunity rather than circulating antibodies. If, however, the

Whether

fungus has succeeded in getting into the tissues, a complex defence response is initiated, the final step of which is killing and digestion. Therefore, other defects in the immune response might be responsible for monilia persisting in human tissues. It has been reported that chronic candida infection can exist in the presence of normal delayed hypersensitivity if there is a deficiency of myeloperoxidase in neutrophils and monocytes.21 In that case the infection is probably due defective clearance function. In other cases chronic candidiasis has been described in association with subnormal lymphocyte transformation due to inhibitory factor in serum 6 as well as abnormally low salivary IgA.3 The frequent occurrence of candida infections in autoimmune and endocrine disorders is well recognised, and varieties of biochemical abnormalities have been reported in patients with chronic moniliasis.6 to a

Our findings therefore underline the fact that chronic candidiasis is a clinical syndrome that can result from various biological deviations, these having the common feature of facilitating monilia organisms to express their parasitic potentialities in humans. Subnormal production of M.I.F. might be an important deviation in this respect. Previous reports 3,4 suggest beneficial effects of lymphoid-cell transplantation in patients with this immune defect, and episodes of graft-versus-host reactions have not been recorded although leucocytes have not always been matched for histocompatibility.4 Our patient has been severely handicapped by infections. We are therefore encouraged to treat her with lymphocytes from her healthy brother, who is a close histocompatibility match. We thank Dr. 1. G. Murray, Dr. H. Buckley, and Dr. J. Faux for serological tests and Mrs. Rose Evans for technical assistance. H. V. is a fellow of the Vigdis and Olafur Memorial Fund in Iceland. for reprints should be addressed to L. H., Departof Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W.12.

Requests

ment

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Chilgren, R. A., Meuwissen, H. J., Quie, P. G., Good, R. A., Hong, R. Lancet, 1969, i, 1268. Canales, L., Middlemas, R. O., Louro, J. M., South, M. A. ibid. 1969, ii, 567. Hermans, P. E., Ulrich, J. A., Markowitz, H. Am. J. Med. 1969, 47, 503. Hobbs, J. R. Broadsheet of the Association of Clinical Pathologists. May, 1970. Baehner, R. L., Nathan, D. E. Science, 1967, 155, 835. Beacon, D. N. J. Lab. clin. Med. 1926, 11, 1092. Brown, R. S., Haynes, H. A., Foley, H. T., Goldwin, H. A., Berard, C. W., Carbone, P. P. Ann. intern. Med. 1967, 67, 291. Holt, L. J., Ling, N. R., Stanworth, D. R. Immunochemistry, 1966, 3, 359. Bach, F. H., Voynow, N. K. Science, 1966, 153, 545. Thor, D. E., Jureziz, R. E., Veach, S. R., Miller, E., Dray, S. Nature, Lond. 1968, 219, 755. Søborg, M. H., Bendixen, G. Acta med. scand. 1967, 181, 247. Carpenter, R. R. J. Immun. 1963, 91, 803. Huber, H., Fudenberg, H. H. Int. Archs Allergy, 1968, 34, 18. Lolekha, S., Dray, S., Gotoff, P. J. Immun. 1970, 104, 296. Winner, H. I. Symposium on Candida Infections; p.6. Edinburgh,

1966. 19. Wilson, J. W. Archs Derm. 1962, 85, 254. 20. Pepys, J. in Clinical Aspects of Immunology (edited by P. L. Gell and R. R. A. Coombs); p. 139. Oxford, 1963. 21. Lehrer, R. I., Cline, M. J. J. clin. Invest. 1969, 48, 1478.