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Delayed maturation of skin window macrophages in myelodysplastic syndromes
Leukemia Research Vol. 13, No. 6, pp. 433-435, 1989. Printed in Great Britain.
0145-2126/89 $3.00 + .00 Maxwell Pergamon Macmillan plc
D E L A Y E D M A T U R A T I O N OF SKIN WINDOW M A C R O P H A G E S IN MYELODYSPLASTIC SYNDROMES H. ZWIERZINA,N. SEPP*,E. RINGLER and F. SCHMALZLt Department of Medicine and *Department of Dermatology, University Innsbruck and t Bezirkskrankenhaus Hall, Austria
(Received 9 December 1988. Accepted 2 January 1989) Abstract--Blood monocyte differentiation to macrophages was examined in nine patients with primary myelodysplastic syndromes using the skin window technique. Emigrated cells were stained cytochemically for acid phosphatase reaction after 1, 2, 4, 7, 9, 12 and 23 h. Compared to age-matched controls, seven patients showed a significant delay in lysosomal enzyme acquisition, which is associated with macrophage differentiation. Our results with this in-vivo assay demonstrate an involvement of the monocyte/macrophage system in primary myelodysplastic syndromes and show that patients often have a disturbance in macrophage differentiation.
Key words: Myelodysplastic syndromes, skin window technique, monocyte-macrophage differentiation, lysosomal enzymes, acid phosphatase.
INTRODUCTION PRIMARY myelodysplastic syndromes represent a clonal disorder of the haematopoietic stem cell which leads to a higher risk for the development of acute nonlymphocytic leukemia [1]. A varying degree of anaemia, leukopenia a n d / o r thrombocytopenia confirms the involvement of all three haematopoietic cell lines [2]. The course of the myelodysplastic syndrome is highly variable with more than 50% of the patients dying from complications related to leukopenia or thrombocytopenia before leukemia develops. Not only the quantity of cells is affected, but also their function, which makes cytopenia even more severe. Functional tests have been applied to neutrophils [3], thrombocytes [4], lymphocytes [5] and erythrocytes [6], and a qualitative affection of all these cell lines was shown in a considerable proportion of patients. The monocyte/macrophage system, however, has rarely been studied in myelodysplastic syndromes. We applied skin w i n d o w t e c h n i q u e to patients with myelodysplastic syndrome to examine macrophage maturation in vivo. Acid phosphatase activity was studied as a marker for lysosomal enzyme content, which is a useful tool for the evaluation of macrophage differentiation [7].
10 --I-~
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I--4--414
"
410
"
120
FIG. 1. Stainless steel instrument which is pressed onto the thigh to stretch the skin (upper view; sizes given in mm).
MATERIALS AND METHODS According to the technique first used by Rebuck and Cowley in 1955 [8], we placed excoriations on the extensor surface of one thigh of seven patients with myelodysplastic syndrome and five age-matched controls. In order to keep the size of the lesion constant, we used a stainless steel instrument (Fig. 1) to stretch the skin. This instrument was pressed onto the extensor surface of the thigh so that the skin was pushed through the hole and thus kept tightly drawn. Different sizes of holes were used to adapt to the nature of the various skin sites. When the instrument was in place, a lesion was made with a sharp cutter (sterile commercially available razor blade).
Correspondence to: Dr Heinz Zwierzina, Universittitsklinik fiir Innere Medizin, A-6020 Innsbruck, Austria. 433
434
H. ZWIERZINAet al. epidermis
razor blade
,
,
papz ~
lagor
FIG. 2. Application of the instrument and the razor blade to the skin.
The excoriation was just deep enough to cut the papillae of the papillary dermis so that punctate bleeding occurred. Sterile cover slides were placed on the excoriations and changed after 1, 2, 4, 7, 9 and 12 h. After 23 h the slides were taken off. The air-dried exudates on slideswith adherent monocytes and neutrophils were stained cytochemically using the acid phosphatase reaction as reported by Barka and Anderson [9].
RESULTS In the first few hours, the emigrating monocytes generally show decreased acid phosphatase activity as compared to blood cells, which can be taken as a sign of degranulation [10]. In preparations which are taken 3-10 h after the skin window has been set up, the mononuclear cells normally show increased enzyme activity near the nucleus and especially in the region of the nuclear hilus. The ferment activity gradually increases until enzymatic activity is more or less distributed throughout the.cytoplasma [11]. In seven out of nine patients with myelodysplastic syndrome we could find a markedly delayed maturation of macrophages in skin window exudates. In all of the seven patients with delayed appearance of enzyme activity, only weak acid phosphatase staining could be seen in the cover slides after 12 h. In two of the seven patients with delayed maturation, after 23 h skin window macrophages showed only little acid phosphatase activity in the paranuclear region. Both these patients had been classified clinically as chronic myelomonocytic leukemia (CMML). Of the five other patients, three had refractory anaemia (RA), and two were classified as refractory anaemia with excess of blasts (RAEB). After 23 h, the slides of these patients showed no diffuse acid phosphatase products throughout the cytoplasm, though paranuclear activity was observed.
DISCUSSION Although there are only few reports of defective in-vitro cell function in primary myelodysplastic syn-
dromes and no results of i n - v i v o tests exist, it seems certain that all haematopoietic cell lines can be functionally affected by various degrees. Increased susceptibility to infection is common even in the absence of neutropenia and contributes essentially to the overall poor prognosis of myelodysplastic syndrome. Neutrophil abnormalities can be recognized morphologically or even better by enzyme-cytochemistry [12]. Monocytes also show abnormal staining patterns in enzyme cytochemistry [12] and aberrant antigen pattern in immunocytology [13], which points to their functional impairment. Although an essential part of the immunologic system, the monocyte/macrophage system has not been extensively studied in myelodysplastic syndromes. Barunsteiner had shown that skin window macrophages are derived from blood cells [14] and Wulff [15] demonstrated that they are derived from monocytes. The functional changes of mononuclear cells in skin window exudates were investigated by Schmalzl using enzyme cytochemical staining [11]. Enzyme cytochemical tests represent the biological status of the cell, and acid phosphatase--a lysosomal enzyme--is a good marker to detect a maturation defect in macrophages [16]. We therefore applied the skin window technique to study the functional abnormalities of the monocyte/ macrophage system in vivo. None of our patients had received cytostatic drugs or was under antibiotic therapy. The present investigation suggests that defects of macrophage maturation are a common feature in patients with myelodyplastic syndromes. This disturbance shows that the involvement of the immunologic system is not restricted to defective neutrophil and lymphocyte function, and gives further reason for the acquisition of severe infections. Further studies are necessary to show possible differences of these functional abnormalities in the monocyte/macrophage system in the different subgroups of myelodysplastic syndromes according to the FAB classification [17].
REFERENCES 1. Galton D. A. G. (1986) The myelodysplastic syndromes. Scand. J. Haemat. 36, Suppl. 45, 11. 2. Abkowitz J., Fialkow P., Niebrugge D., Raskind W. & Adamson J. (1984) Pancytopenia as a clonal disorder of a multipotent stem cell. J. clin. Invest. 73, 258. 3. Martin S., Baldock S., Ghomein A. & Child J. (1983) Defective neutrophil function and microbicidal mechanism in the myelodysplastic disorders. J. clin. Path. 36, 1120. 4. Lintula R., Rasi V., Ikkala E., Borgstr6m G. & Vuopio
Maturation of skin window macrophages P. (1981) Platelet function in preleukemia. Scand. J. Haemat. 26, 65. 5. Porzolt F. & Heimpel H. (1982) Natural killer cell activity in preleukemia. Lancet i, 449. 6. Lintula R. (1986) Ferrokinetic abnormalities and red cell life span in myelodysplastic syndromes: a review. Scand. J. Haemat. 36, Suppl. 45, 48. 7. Cohn Z., Fedorko M. & Hirsche J. (1966) The in-vitro differentiation of mononuclear macrophages. V. The formation of macrophage lysosomes. J. exp. Med. 123, 757. 8. Rebuck J. & Cowley J. (1955) A method of studying leukocytic function in vivo. Ann. N.Y. Acad. Sei. 59, 757. 9. Barka T. & Anderson P. (1962) Histochemical method for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10, 741. 10. Schmalzl F., Wiedermann C. & Braunsteiner H. (1982) Quantitativ-zytochemische Untersuchung der SaurePhosphatase-Aktivit~it in Hautfenster-Makrophagen. Acta med. austri. 9, 29. 11. Schmalzl F., Huber H., Asamer H., Abbrederis K. & Braunsteiner H. (1969) Cytochemical and immunohistologic investigations on the source and the func-
435
tional changes of mononuclear cells in skin window exudates. Blood 34, 129. 12. Schmalzl F., Konwalinka G., Michlmayr G., Abbrederis K. & Braunsteiner H. (1978) Detection of cytochemical and morphological anomalies in preleukemia Acta haemat. 59, 1. 13. Clark R. & Jakobs A. (1985) Granulocyte and monocyte surface membrane markers in the myelodysplastic syndromes. J. clin. Path. 38, 301. 14. Braunsteiner H. (1961) Exp6riences de marquage des lymphocytes par la thymidine triti6e. Nouv. Revue ft. H~mat. 1, 733. 15. Wulff H. (1963) Histochemical studies of leukocytes from an inflammatory exudate. V. Alkaline and acid phosphatases and esterases. Acta haemat. 30, 159. 16. Schmalzl F. & Braunsteiner H. (1970) The cytochemistry of monocytes and macrophages. Ser. Haemat. 3, 93. 17. Bennett J., Catovsky D., Flandrin G., Daniel M. T., Galton D. A. G., Gralnick H. & Sultan C. (1982) The French-American-British (FAB) Co-operative Group: proposals for the classification of the myelodysplastic syndromes. Br. J. Haernat. 1, 189.
0145-2126/89 $3.00 + .00 Maxwell Pergamon Macmillan plc
D E L A Y E D M A T U R A T I O N OF SKIN WINDOW M A C R O P H A G E S IN MYELODYSPLASTIC SYNDROMES H. ZWIERZINA,N. SEPP*,E. RINGLER and F. SCHMALZLt Department of Medicine and *Department of Dermatology, University Innsbruck and t Bezirkskrankenhaus Hall, Austria
(Received 9 December 1988. Accepted 2 January 1989) Abstract--Blood monocyte differentiation to macrophages was examined in nine patients with primary myelodysplastic syndromes using the skin window technique. Emigrated cells were stained cytochemically for acid phosphatase reaction after 1, 2, 4, 7, 9, 12 and 23 h. Compared to age-matched controls, seven patients showed a significant delay in lysosomal enzyme acquisition, which is associated with macrophage differentiation. Our results with this in-vivo assay demonstrate an involvement of the monocyte/macrophage system in primary myelodysplastic syndromes and show that patients often have a disturbance in macrophage differentiation.
Key words: Myelodysplastic syndromes, skin window technique, monocyte-macrophage differentiation, lysosomal enzymes, acid phosphatase.
INTRODUCTION PRIMARY myelodysplastic syndromes represent a clonal disorder of the haematopoietic stem cell which leads to a higher risk for the development of acute nonlymphocytic leukemia [1]. A varying degree of anaemia, leukopenia a n d / o r thrombocytopenia confirms the involvement of all three haematopoietic cell lines [2]. The course of the myelodysplastic syndrome is highly variable with more than 50% of the patients dying from complications related to leukopenia or thrombocytopenia before leukemia develops. Not only the quantity of cells is affected, but also their function, which makes cytopenia even more severe. Functional tests have been applied to neutrophils [3], thrombocytes [4], lymphocytes [5] and erythrocytes [6], and a qualitative affection of all these cell lines was shown in a considerable proportion of patients. The monocyte/macrophage system, however, has rarely been studied in myelodysplastic syndromes. We applied skin w i n d o w t e c h n i q u e to patients with myelodysplastic syndrome to examine macrophage maturation in vivo. Acid phosphatase activity was studied as a marker for lysosomal enzyme content, which is a useful tool for the evaluation of macrophage differentiation [7].
10 --I-~
[
I--4--414
"
410
"
120
FIG. 1. Stainless steel instrument which is pressed onto the thigh to stretch the skin (upper view; sizes given in mm).
MATERIALS AND METHODS According to the technique first used by Rebuck and Cowley in 1955 [8], we placed excoriations on the extensor surface of one thigh of seven patients with myelodysplastic syndrome and five age-matched controls. In order to keep the size of the lesion constant, we used a stainless steel instrument (Fig. 1) to stretch the skin. This instrument was pressed onto the extensor surface of the thigh so that the skin was pushed through the hole and thus kept tightly drawn. Different sizes of holes were used to adapt to the nature of the various skin sites. When the instrument was in place, a lesion was made with a sharp cutter (sterile commercially available razor blade).
Correspondence to: Dr Heinz Zwierzina, Universittitsklinik fiir Innere Medizin, A-6020 Innsbruck, Austria. 433
434
H. ZWIERZINAet al. epidermis
razor blade
,
,
papz ~
lagor
FIG. 2. Application of the instrument and the razor blade to the skin.
The excoriation was just deep enough to cut the papillae of the papillary dermis so that punctate bleeding occurred. Sterile cover slides were placed on the excoriations and changed after 1, 2, 4, 7, 9 and 12 h. After 23 h the slides were taken off. The air-dried exudates on slideswith adherent monocytes and neutrophils were stained cytochemically using the acid phosphatase reaction as reported by Barka and Anderson [9].
RESULTS In the first few hours, the emigrating monocytes generally show decreased acid phosphatase activity as compared to blood cells, which can be taken as a sign of degranulation [10]. In preparations which are taken 3-10 h after the skin window has been set up, the mononuclear cells normally show increased enzyme activity near the nucleus and especially in the region of the nuclear hilus. The ferment activity gradually increases until enzymatic activity is more or less distributed throughout the.cytoplasma [11]. In seven out of nine patients with myelodysplastic syndrome we could find a markedly delayed maturation of macrophages in skin window exudates. In all of the seven patients with delayed appearance of enzyme activity, only weak acid phosphatase staining could be seen in the cover slides after 12 h. In two of the seven patients with delayed maturation, after 23 h skin window macrophages showed only little acid phosphatase activity in the paranuclear region. Both these patients had been classified clinically as chronic myelomonocytic leukemia (CMML). Of the five other patients, three had refractory anaemia (RA), and two were classified as refractory anaemia with excess of blasts (RAEB). After 23 h, the slides of these patients showed no diffuse acid phosphatase products throughout the cytoplasm, though paranuclear activity was observed.
DISCUSSION Although there are only few reports of defective in-vitro cell function in primary myelodysplastic syn-
dromes and no results of i n - v i v o tests exist, it seems certain that all haematopoietic cell lines can be functionally affected by various degrees. Increased susceptibility to infection is common even in the absence of neutropenia and contributes essentially to the overall poor prognosis of myelodysplastic syndrome. Neutrophil abnormalities can be recognized morphologically or even better by enzyme-cytochemistry [12]. Monocytes also show abnormal staining patterns in enzyme cytochemistry [12] and aberrant antigen pattern in immunocytology [13], which points to their functional impairment. Although an essential part of the immunologic system, the monocyte/macrophage system has not been extensively studied in myelodysplastic syndromes. Barunsteiner had shown that skin window macrophages are derived from blood cells [14] and Wulff [15] demonstrated that they are derived from monocytes. The functional changes of mononuclear cells in skin window exudates were investigated by Schmalzl using enzyme cytochemical staining [11]. Enzyme cytochemical tests represent the biological status of the cell, and acid phosphatase--a lysosomal enzyme--is a good marker to detect a maturation defect in macrophages [16]. We therefore applied the skin window technique to study the functional abnormalities of the monocyte/ macrophage system in vivo. None of our patients had received cytostatic drugs or was under antibiotic therapy. The present investigation suggests that defects of macrophage maturation are a common feature in patients with myelodyplastic syndromes. This disturbance shows that the involvement of the immunologic system is not restricted to defective neutrophil and lymphocyte function, and gives further reason for the acquisition of severe infections. Further studies are necessary to show possible differences of these functional abnormalities in the monocyte/macrophage system in the different subgroups of myelodysplastic syndromes according to the FAB classification [17].
REFERENCES 1. Galton D. A. G. (1986) The myelodysplastic syndromes. Scand. J. Haemat. 36, Suppl. 45, 11. 2. Abkowitz J., Fialkow P., Niebrugge D., Raskind W. & Adamson J. (1984) Pancytopenia as a clonal disorder of a multipotent stem cell. J. clin. Invest. 73, 258. 3. Martin S., Baldock S., Ghomein A. & Child J. (1983) Defective neutrophil function and microbicidal mechanism in the myelodysplastic disorders. J. clin. Path. 36, 1120. 4. Lintula R., Rasi V., Ikkala E., Borgstr6m G. & Vuopio
Maturation of skin window macrophages P. (1981) Platelet function in preleukemia. Scand. J. Haemat. 26, 65. 5. Porzolt F. & Heimpel H. (1982) Natural killer cell activity in preleukemia. Lancet i, 449. 6. Lintula R. (1986) Ferrokinetic abnormalities and red cell life span in myelodysplastic syndromes: a review. Scand. J. Haemat. 36, Suppl. 45, 48. 7. Cohn Z., Fedorko M. & Hirsche J. (1966) The in-vitro differentiation of mononuclear macrophages. V. The formation of macrophage lysosomes. J. exp. Med. 123, 757. 8. Rebuck J. & Cowley J. (1955) A method of studying leukocytic function in vivo. Ann. N.Y. Acad. Sei. 59, 757. 9. Barka T. & Anderson P. (1962) Histochemical method for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10, 741. 10. Schmalzl F., Wiedermann C. & Braunsteiner H. (1982) Quantitativ-zytochemische Untersuchung der SaurePhosphatase-Aktivit~it in Hautfenster-Makrophagen. Acta med. austri. 9, 29. 11. Schmalzl F., Huber H., Asamer H., Abbrederis K. & Braunsteiner H. (1969) Cytochemical and immunohistologic investigations on the source and the func-
435
tional changes of mononuclear cells in skin window exudates. Blood 34, 129. 12. Schmalzl F., Konwalinka G., Michlmayr G., Abbrederis K. & Braunsteiner H. (1978) Detection of cytochemical and morphological anomalies in preleukemia Acta haemat. 59, 1. 13. Clark R. & Jakobs A. (1985) Granulocyte and monocyte surface membrane markers in the myelodysplastic syndromes. J. clin. Path. 38, 301. 14. Braunsteiner H. (1961) Exp6riences de marquage des lymphocytes par la thymidine triti6e. Nouv. Revue ft. H~mat. 1, 733. 15. Wulff H. (1963) Histochemical studies of leukocytes from an inflammatory exudate. V. Alkaline and acid phosphatases and esterases. Acta haemat. 30, 159. 16. Schmalzl F. & Braunsteiner H. (1970) The cytochemistry of monocytes and macrophages. Ser. Haemat. 3, 93. 17. Bennett J., Catovsky D., Flandrin G., Daniel M. T., Galton D. A. G., Gralnick H. & Sultan C. (1982) The French-American-British (FAB) Co-operative Group: proposals for the classification of the myelodysplastic syndromes. Br. J. Haernat. 1, 189.