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Arch. Derm. Res. 257, 157-161 (1976)



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Absence of Leukocyte-Migration Inhibitory Factor in Serum from Patients with Mycosis Fungoides Henning Thulin Department of Dermatology and Venerology, Marselisborg Hospital, University of Aarhus, DK-8000 Aarhus, Denmark

Summary. Sera from 16 patients with mycosis fungoides at various stages of the disease were investigated for the presence of a leukocyte-migration inhibitory factor. The agarose gel technique with human peripheral blood leukocytes as migating indicator cells was used for the purpose. The migration inhibitory effect of patients sera, pooled AB-sera and sera from control persons was tested on peripheral blood leukocytes from patients and controls. Also upconcentrated sera were used. The presence of substances with migration inhibitory effect in serum from patients with mycosis fungoides could not be demonstrated. The significance of these findings is discussed.

Zusammenfassung. Das Serum von 16 Patienten mit Mykosis fungoides in verschiedenen Entwicklungsstadien wurde auf das Vortiegen des Leukocytenmigrations-Inhibitor-Faktors untersucht. Die Agarose-Gel-Technik mit menschlichen Leukocyten aus dem Blut als Indikatorzelle wurde methodisch verwandt. Der Leukocyten-migrationshemmende Effekt von AB-Sera und Sera yon Kontrollpersonen wurd an peripheren Blutleukocyten der Patienten und Kontrollen getestet. H6her konzentrierte Sera wurden ebenfalls verwandt. Im Serum yon Patienten mit Mykosis fungoides konnten keine Substanzen aufgefunden werden, die einen Leukocyten-migrationshemmenden Effekt besaBen. Introduction Several recent studies indicate the presence of MIF-like (migration inhibitory factor) activity in serum from patients with certain lymphoprolipherative diseases [3] and in serum from patients with Sezary syndrome [14]. Investigations in mycosis fungoides should be of special interest due to the close relationship to Sezary syndrome. Both have been proposed to be T-cell malignancies [6]. The presence of malignant T-cells might produce a variety of lymphokines in mycosis fungoides. *

This work was supported by a grant from the P. Carl Petersens Foundation


H. Thulin

In t h e p r e s e n t s t u d y s e r u m f r o m p a t i e n t s w i t h m y c o s i s f u n g o i d e s at v a r i o u s s t a g e s of t h e d i s e a s e was t e s t e d for M I F - l i k e activity, u s i n g l e u k o c y t e m i g r a t i o n a g a r o s e t e c h n i q u e [2].

Materials and Methods A total of 16 patients with mycosis fungoides at various stages of the disease were investigated. Staging was performed in accordance with the classification by Van Scott [ 11]. Two patients were in stage IV. 4 in stage III, 7 in stage II and 3 in stage I. In 7 patients and 7 control persons, matched for age and sex, test were performed to evaluate the influence of autologous serum, AB-serum and serum from the matched control or mycosis patient respectively on the migration of own peripheral blood leukoeytes. Controls were suffering from minor dermatological disorders. They received no systemic medicamentation, nor topical steroids. Serum was heat-inactivated at 56 ° C for 30 rain. Cultures were set up at the same time for a patient and the matched control. Serum from 13 patients with mycosis fungoides were tested using peripheral blood leukocytes from 13 normal persons as indicator dells. All controls had blood type 0. The influence on cell migration of mycosis-serum was compared to that of pooled AB-serum. Unconcentrated serum and serum upconcentrated 5 times were used. All sera were freezedryed and stored until use (stored up to 1 month). Just before use sterile water was added in adequate volumina. Serum obtained from patients and controls and AB-serum from healthy blood donors was heat inactivated at 56°C for 30 min. In a preliminary study, prior to this investigation, the influence of pooled AB-serum on the migration of normal peripheral blood leukocytes was tested. The inhibitory effect of AB-serum compared to TC medium was not increased by upconcentration (mean: 0.93 and 1.09 respectively), on the contrary a stimulatory effect could be recorded. An agarose gel technique was used in accordance with the technique described by Clausen [2]. Peripheral blood leukocytes were suspended in TC medium 199 (Difco) with 10% horse serum and penicillin/streptomycin added. Serum from patients, from controls or pooled AB-serum was added to the medium in a concentration up to 50%. Six cultures were set up in each group. Incubation was carried out for 1.5 h. Results were expressed as a ratio between the average migration area from cul-

Table 1. Influence of autologous serum, AB-serum and serum from matched control or mycosis patient on the migration of own peripheral blood leukocytes. Ratio between average area from cultures with different sera and cultures with tissue culture medium (TC 199, Difco), appears. No difference was found between mycosis patients and controls Ratio homologous, autologous,or AB-serum/TC medium

Mean S.D.

Patients with mycosis fungoides

Control group







0.84 0.93 0.93 0.98 0.89 1.04 0.80

0.88 0.89 0.83 1.07 0.98 1.47 0.86

0,83 0.93 0.88 1.08 0.93 1.24 0,76

0.88 0.87 0.89 0.79 1.09 0.96 0.68

0.84 0.87 0.91 0.73 1.18 1.07 0.85

0.91 0.84 0.96 0.74 1.25 1.13 0.78

0.92 0.1i

1.00 0.22

0.95 0.14

0.88 0.14

0.92 0.14

0.94 0.17

Leukocyte-Migration Inhibitory Factor in Serum


Table 2. Influence of unconcentrated and 5 times concentrated serum from patients with mycosis fungoides on the migration of normal peripheral blood leukocytes. Ratio between average migration are from cultures medium (TC i99. Difco) is recorded. No migration inhibition of normal peripheral blood leukocytes, was induced by mycosis serum Ratio mycosis serum/AB-serum in control persons tested with mycosis serum Unconcentrated serum 0.86 0.9a 1.04 0.94 1.15 0.95 0.97 0.99 1.10 0.99 [.03 1.14 1.12 Mean S.D.

1.02 0.11

Concentrated serum × 5 0.89 0.99 1.09 1.07 1.01 0.97 1.02 1.04 1.00 1.10 1.27 1.04 0.1I

tures containing TC medium or as a ratio between the average migration area from cultures with mycosis serum and the average migration area from cultures with AB-serum.

Results Table 1 shows results from testing A B - s e r u m , autologous and homologous serum on patients and controls peripheral blood leukocytes. The migration inhibitory effect of autologous serum was c o m p a r e d to that of A B - s e r u m . Neither in patients nor in controls did autologous serum inhibit the migration. In the same trial mycosisserum was tested on controls leukocytes and control-serum on patients leukocytes. Mycosis-serum did not inhibit the migration of control leukocytes c o m p a r e d to A B serum. No inhibitory effect of control-serum on the migration of leukocytes from the matched mycosis patients could be seen. The stage of the disease was of no importance in this aspect, The influence of mycosis serum on the migration of leukocytes from normal blood donors, blood type 0, c o m p a r e d to A B - s e r u m , appears from Table 2. No significant difference could be d e m o n s t r a t e d between the effect of mycosis-serum and A B - s e r u m . N o difference was found between ratio in c o n c e n t r a t e d serum c o m p a r e d to ratio in unconcentrated serum.

Discussion The presence of factors with M I F - l i k e activity in tissue fluids has been described in a n u m b e r of recent studies. M I F - l i k e activity has been d e m o n s t r a t e d in joint fluid from rabbits with experimental arthritis [9], in serum from immunized mice


H. Thulin

and guinea pigs after challenge [8, 12] and in peritoneal exudates from immunized guinea pigs [13]. Also in certain human pathological states lymphokines might be detectable. Macrophage migration inhibitory activity could be detected in a majority of patients with Iymphoprolipherative diseases: chronic lymphocytic leukemia. Hodgkins disease, lymphoma and myeloma [3]. Serum from children in remission from acute lymphoblastic leukemia could inhibit the migration of buffy coat cells from normal children [10]. In a serum assay for migration inhibitory factor Yoshida [14] demonstrated the presence of a factor with MIF-like activity in patients with Sezary syndrome. Sezary syndrome is caracterized by abnormal cells showing T cell properties [4,5,15]. Sezary syndrome is considered a leukemic variant of mycosis fungoides. Both diseases are T cell malignancies (for a review: 6). Since T cells are known to produce lymphokines it could be expected that in a disease with malignant T-cells an increased amount of lymphokines could be recorded. Although this was the case in Sezary syndrome [14], we were not able to demonstrate a similar presence of factors in serum from patients with mycosis fungoides with migration inhibitory effect on human buffy coat cells. This is in accordance with Yoshida who tested some mycosis patients, but found no inhibitory activity by a serum assay [14]. The reasons for not finding a migration inhibitory factor in serum from patients with mycosis fungoides could be several. The factor could be absent or the concentration to low to measure due to the few abnormal T cells producing the factor compared to the larger number of cells in Sezary syndrome. Rocklin [7] found two distinct migration inhibition factors in supernatants from antigen-stimulated human lymphocytes: MIF mot.-weight 23000 released from both B and T cells detectable by guinea pig macrophages and LIF mol.-weight 69000 detectable by human leukocytes, prevalently a T-lymphocyte. product. It has been shown by Bendtzen [1] that LIF can be tested in an agarose technique using peripheral blood leukocytes as migratory cells. MIF could be present without LIF giving no migration inhibition of buffy coat cells, but of guinea pig macrophages. One would expect the presence of LIF in mycosis fungoides being a T cell lymphoma [6]. LIF activity has been shown to disappear during storage [1] however we investigated fresh serum. In conclusion, according to the study by Yoshida and our study, neither MIF nor LIF seem to be present in measurable amounts in the serum from patients with mycosis fungoides.

References I. Bendtzen, K., Andersen. V., Bendixen, G.: An in vitro assay of leukocyte migration inhibitor), activity from human lymphocytes stimulated with concanavalinA. Acta allerg. 30, 133-149 (1975) 2. Clausen, J. E.: Migration inhibitory,effect of cell-free supernatant from tuberculin-stimulated cultures of human mononuclear leukocytes demonstrated by two-step MIF agarose assay. J. Immun. 110, 546--551 (1973) 3. Cohen, S.: Serum migration-inhibitory activity in patients with lymphoproliferative diseases. New Engl. J. Med. 290, 882-886 (1974) 4. Edelson, R. L.: Morphologic and functional properties of the atypical T-lymphocytes of the Sezary syndrome. Mayo Clinic Proc. 49, 559-566 (1974) 5, Flandrian, G., Brouet, J. C.: The Sezary cell. Cytologic,cytochemicaland immunologicstudies. Mayo Clinic Proc. 49, 575-583 (1974) 6, Lutzner. M.:CutaneousT-celllymphomas. The Sezarysyndrome,mycosisfungoides,andrelated disorders. Ann. int. Med. 83, 534-552 (1975)

Leukocyte-Migration Inhibitory, Factor in Serum


7. Rocklin, R. E.: Products of activated lymphocytes. Leukocyte inhibitory factor (L[F) distinct from migration inhibitory, factor (MIF). J. Immun. 112, 1461-1466 (1974) 8. Salvin. S. B.. Younger, J. S., Lederer, L. H.: Migration inhibitory, factor and interferon in the circulation of mice with delayed hypersensitivity. Infect. Immun. 7, 68-75 (1973) 9. Stastny, P., Cooke, T. D., Ziff, M.: Production of a macrophage migration inhibitory factor in rabbits with experimental arthritis. Clin. exp. Immun. 14, 141-147 (1973) 10. Szigeti, R., R6v6sz, R., Ger6-Ferencz. E.: The inhibitory, effect of leukaemia associated antigen and leukaemia serum on the leukocyte migration of children with acute leukaemia in remission. Acta allerg. 29, 288-296 (1974) 11. Scott. E. J.. van, Kalmanson, J. D.: Complete remissions of mycosis fungoides lymphoma induced by topical nitrogen mustard (HN2). Cancer 32, 18-30 (1973) 12. Yamamoto, K., Takahashi, Y.: Macrophage migration inhibition by serum from desensitized animals previously sensitized with tubercle bacilli. Nature (Lond.) 233, 261-263 (1971) 13. Yoshida, T., Cohen, S.: In vivo manifestations of lymphokine and lymphokine activity. Mechanism of cell-mediated immunity, p. 43. Ed, McCluskey, R. T., Cohen, S. New York: J. Wiley and Sons 1974 14. Yoshida, T.: Migration inhibitory activity in serum and cell supernatants in patients with Sezary syndrome, J. Immun. 114, 915-918 (1975) 15. Zucker-Franklin, D.: Properties of Sezary lymphoid cell. Mayo Clinic Proc. 49, 567-574 (1974) Received August 18, 1976

Absence of leukocyte-migration inhibitory factor in serum from patients with mycosis fungoides.

Archives for Arch. Derm. Res. 257, 157-161 (1976) erma .logicai search © by Springer-Verlag 1976 Absence of Leukocyte-Migration Inhibitory Factor...
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