Chronic myelogenous leukemia: Effect of interferon-α treatment on phagocytic activity and capacity of circulating neutrophils

Leukemra Research Vol. 22, No. 2. pp 1 IS- 117. 1998. D 1998 Ekewer Science Ltd All rights reserved Pnnled m Great Breun 0145s2126/98 $19.00 + 000

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CHRONIC MYELOGENOUS LEUKEMIA: EFFECT OF INTERFERON-a TREATMENT ON PHAGOCYTIC ACTIVITY AND CAPACITY OF CIRCULATING NEUTROPHILS Sabine Kasimir-Batter*, Hellmut Ottingert, Marc Wuttke*, Werner Stellberg”, Hans Grosse-Wilde?, Siegfried Seeber* and Max Ernst Scheulen” *Department of Internal Medicine (Cancer Research), University of Essen Medical School, HufelandstralJe 55,45 122 Essen, Germany and tInstitute of Immunology, University of Essen Medical School, 45 122 Essen, Germany

(Received 3 March 1997. Accepted 14 June 1997) Abstract-This

study focuses on the effect of interferon (IFNI-c( on phagocytosis of FITC-labeled neutrophils (PMNs) in chronic myelogenous leukemia (CML). The phagocytic activity and capacity of PMNs from IFN-CI treated patients (n= 171, untreated CML patients (n = 9) and from healthy donors (n= 20) were compared using flow cytometry. Both parameters of PMN phagocytosis were reduced in untreated CML and in IFN-c( treated CML with Phl chromosome persistence but normal in IFN-a treated CML with Phl conversion. Thus, the phagocytic performance of PMNs in patients with chronic phase CML is significantly improved after successful treatment with IFN-3. ,Q 1998 Elsevier Science Ltd. All rights reserved.

Escherichia co/i by polymorphonuclear

Key words: chronic phils, phagocytosis.

myelogenous

leukemia,

interferon

Introduction

(IFN)-s(, polymorphonuclear

neutro-

male, 11 female) aged 17-73 years (median 52 years) with definite diagnosis of first chronic phase Ph’+CML. Patient characteristics at the time of study entry are summarized in Table 1. Nine patients were enrolled at the time of diagnosis (no treatment group); all of them received either IFN-cr or hydroxyurea later. Seventeen patients were under IFN-a at a dosage of 3-5 million units three times weekly for >3 months when studied (treatment group) including three patients with complete cytogenetic remission and 14 patients with persisting Ph’ chromosome. In 10 patients of the latter group leukocyte counts had returned to the normal range. Patients Nos 1 and 2 of Table 1 exhibited markedly elevated leukocyte counts after reduction of IFN-c( from 5 to 3 million units three times per week because of drug side-effects (increase of bone pain). Controls were 20 healthy volunteers aged 23-73 years. Isolation of Ph4Ns. PMNs were isolated from 40 ml heparinized blood (15 U/ml) using PolymorphoprepO (Nycomed, Oslo, Norway). After lysis of erythrocytes the PMN cell fraction was suspended in phosphatebuffered saline (PBS) to a final concentration of 1 x lo7 cells/ml. Isolated cells underwent further analysis if the percentage of neutrophil PMNs was >95% and contamination with mononuclear myeloid cells was

It is well known that polymorphonuclear neutrophils (PMNs) from chronic myelogenous leukemia (CML) patients display defective phagocytosis [l] and diverse functional alterations on a biochemical level [2-51. Studies on the effect of interferon (IFN)-c( on phagocytosis are sparse, but IFN-cr has been reported to enhance some biochemical alterations of CML-PMNs in viva and in vitro [5]. Since the biological relevance of these findings is unknown we studied the ingestion of opsonized fluorescein (FITC)-labeled Escherichiu coli by PMNs from IFN-cr treated patients as compared to untreated CML and healthy donors. Results were expressed in terms of phagocytic activity (percentage of PMNs ingesting at all) and phagocytic capacity (amount of material ingested per cell). Patients and methods Patients The present study enrolled 26 adult Caucasians (15 Correspondence to: Department of Internal Medicine (Cancer Research), West German Cancer Centre Essen, University of Essen Medical School, HufelandstraRe 5.5, 4.5 122 Essen, Germany [Tel. (+49) 201 723 3112; Fax (+49) 201 723 37901. 115

.7 -r

S. Kasimir-Bauer

116

Table 1. Patient characteristics at entry into study No

Sex

Age

IFN-a Treated CML 44 1 m 2 3

f

7

m f f m m

8

m

9

f m m m

4 5 6

10 11 12

13

f

14

m f m f

15 16 17

Untreated CML 18 f 19 m 20 m 21 m 22 23 24 25 26

f

m f f m

33 68 50 48 70 68 50 73 6.5 38 71 43 25 52 71 71

18 57 59 59 52 48 45 41 23

WBC*

ph’

c-abl-bcr

n

9.4 9.4 9.3 9.2 6.7 6.2 5.8 5.4 5.2 5.2 5 4.3 3.4

+ + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + +

124.2 61.5 55.2 36.5 28.8 28.2 22.0 16.8 15.7

+ + + + + + + + +

+ + + + + + + + +

50.6 31.5

11.8 10.2

.

+ 0 Controls

IFN + (Ph -1

IFN + (Ph +)

IFN (Ph +I

n = 20

II = 3

n = 14

tl=9

Fig. 1. Phagocytic activity of PMNs (percentage of PMNs ingesting at all) in chronic phase Ph’+ CML and in healthy controls. IFN-a+, CML treatedwith IFN-a; IFN-a-, untreated CML; Ph-, complete cytogenetic remission; Ph+, persistence of the Ph’ chromosome;n, number of patients. Isolated PMNs (1 x 106) were incubated with 20 pl of FITC-labeled opsonized E. coli (1 x 109/ml) for 10 min at 37°C. The data are given by combined scatter column/box-plot diagrams. Boxes extend from the 25th-75th percentiles,with a horizontal line at the median. Whiskers go down to the smallest and up to the largest value. **P < 0.01 and ***P < 0.001, when compared to controls.

Results < 1% (Pappenheim-stained smears, differential cell counts). Evaluation of PMN phagocytosis. PMN phagocytosis was determined by flow cytometry using Phagotest@ (Orpegen, Heidelberg, Germany). In brief, 1 x lo6 isolated PMNs/lOO pl were incubated with 20 pl of precooled FITC-labeled opsonized E. coli bacteria (1 x lO”/ml) for 10 min at 37”C, washed twice (5 min, 25Og, 4”C), incubated with 100 pl of the DNA-staining solution for 10 min at 4°C before intracellular fluorescence was determined in 15 000 cells per sample using an EPICS XL flow cytometer (Coulter, Krefeld, Germany). Phagocytosis was quantified in terms of phagocytic activity (percentage of PMNs ingesting at all, i.e. uptake of 3 1 bacterium per cell) and phagocytic activity (i.e. the intensity of intracellular fluorescence which is correlated to the number of bacteria ingested per cell). Fluorescence intensity was expressed as channel of mean fluorescence intensity (CMFI) based on the number of events in channel O-1023. Statistical analysis. One-way analysis of variance (one-way ANOVA) followed by Bonferroni’s post test for multiple comparisons was used.

The phagocytic activity The phagocytic activity (mean? S.D.) of PMNs (cf Fig. 1) was found to be significantly lower in untreated CML (56 + 13%; P < 0.001) and in IFN-a treated CML (71 +_ 23%; P < 0.01) as compared to healthy controls (89 + 9%). When analysis was restricted to IFN-a treated CML with Phl persistence the mean phagocytic activity was 67 k 23% (P < 0.01 as compared to normal controls, but N.S. as compared to untreated CML). Interestingly, PMNs from patients who responded worst to IFN-c( (Table 1, patients Nos l-3) had the lowest phagocytic activities (28.3, 34.3 and 36%, respectively). In contrast, PMNs from all three patients with Phl conversion (Table 1, patients Nos 1517) exhibited phagocytic activities within the range of healthy donors (91, 8 1 and 94%, respectively). Phagocytic capacity Phagocytic capacity (mean+S.D.) of PMNs (cf Fig. 2) was also found to be significantly lower in untreated (389 _+ 118 CMFI; P < 0.04) and in IFN-a treated CML (424 f 239 CMFI; P < 0.05) as compared to healthy controls (609 + 161 CMFI) (Fig. 2). In IFN-a treated CML with Phl persistence the mean phagocytic

BCR/ABL 1000

**

r .l-

*

! l

Controls n = 20

IFN + (Ph -)

IFN + W’h +)

IFN W’h +)

n=3

n = 14

n=9

Fig. 2. Phagocytic capacity of PMNs (quantity of bacteria ingested per cell) in chronic phase Ph’+ CML and in healthy controls. The intensity of incorporated fluorescence was measuredin 15 000 PMNs of each sample. Results for each sample were expressed as CMFI, with O-1023 channels available. *P < 0.05, **P < 0.01. For further details see legend to Fig. 1.

capacity was 358 k 206 CMFI (P < 0.01 as compared to healthy donors, but N.S. as compared to untreated CML). In the three patients with Phl conversion (Table 1, patients Nos 15-17) the phagocytic activities were normal (902, 612 and 550 CMFI, respectively). Discussion This study shows that PMNs from untreated CML patients are defective in terms of phagocytic activity and phagocytic capacity and hereby extends previous findings [l]. These data suggest that circulating PMNs arising from the Ph ‘+ clone ingest E. coZi less efficiently than do normal neutrophils since: (1)

(2)

Malignant PMNs are known to be well represented in the circulation in untreated CML [6]; and Contamination of our PMN samples by basophils and immature neutrophils was too low ( < 5%) to account for the markedly reduced phagocytosis observed in CML.

We cannot exclude that the PMNs from CML patients may be less mature than PMNs from normal donors. However, if so, this lack of maturity is an integral aspect of CML pathophysiology. Another finding of this study is that the phagocytic activity and capacity of PMNs did not differ significantly in untreated and IFN-E treated

117

and leukemia

CML. Notably, however, the scatter for both parameters of phagocytosis was much larger in the IFN-a treated group. We suggest that the more successful IFN-IX treatment is in reducing the Ph’+ clone the more normal PMN phagocytosis becomes in CML since firstly, phagocytosis was found to be normal only in those three EN-a patients who had achieved a complete cytogenetic remission and secondly, phagocytic activity was lowest in the three patients of the study in whom IFN-c( clearly failed to induce a hematological remission. In any way a direct effect of EN-a on phagocytic activity and capacity of CML PMNs can be excluded as evidenced by in vitro priming (dose and time kinetics) of untreated CML PMNs with IFN-cr (data not shown). There are previous data from our group and others indicating that some functional alterations are induced by IFN-cr treatment on the biochemical level in normal and CML PMNs [5,7,8]. However, in view of our present data we feel that the observed biochemical alterations are of minor relevance since ongoing IFN-a treatment did not reduce phagocytic activity or capacity in our patients with Ph’ conversion. Acknowledgements-This work was supported by Wrderverein Essener Tumorklinik e.V. For editorial assistance we are indebted to Mrs Ch. Wartchow.

References 1. Carulli, G., The neutrophil granulocyte in chronic myeloid leukemia. Haematologia, 1993, 25, 35. 2. Kaplan, S. S., Berkow, R. L., Joyce, R. A., Basford, R. E. and Barton, J. C., Neutrophil function in chronic neutrophilic leukemia: defective respiratory burst in response to phorbol esters. Acta Haematologica, 1992, 87, 16.

3. Kant, A., Advani, S. and Zingde, S., FcgRII and FcgRIII on normal and leukaernic granulocytes: A flow cytometry and northern analysis. Leukemia Research, 1996, 20, 93. 4. Carulli, G., Gianfaldoni, M. L., Azzara, A., Papineschi, F., Vanacore, R., Minucci, S., Testi, R. and Ambrogi, F., FcRIII (CDl6)expression on neutrophils from chronic myeloid leukemia. A flow cytometric study. Leukemia Research, 1992, 16, 1203. 5. Kasimir-Bauer, S., Ottinger, H., Brittinger, G. and K6nig, W., Philadelphia chromosome-positive chronic myelogenous leukemia: functional defects in circulating mature neutrophils of untreated and interferon-u-treated patients. Experimental Hematology, 1994, 22, 426. 6. Gricochea, B., Chase, A., Lazaridou, T. and Goldman, J. M., T lymphocytes in chronic myelogenous leukemia (CML): evidence of the bcr-abl fusion gene detected by fluorescence in situ hybridization in 14 patients. Leukemia, 1994,8, 1197. 7. Kasimir, S., Brom, J. and KGnig, W., Effect of interferon alpha on neutrophil functions. Immunology, 1991, 74, 271. 8. Melby, K., Midtvedt, T. and Degre, M., Effect of human leukocyte interferon on phagocytic activity of polymorphonuclear leukocytes. Acta Pathologica Microbiologica Immunologica Scandinavia (B), 1982, 90, 181.