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Irradiation and monocyte function in patients with breast cancer
036&3016/79/112063-05$02.00/O
Int. .I. Radiation Oncology Biol. Phys., Vol. 5, pp. 2063-2067 o Pergamon Press Ltd., 1979. Printed in the U.S.A.
??Brief Communication
IRRADIATION AND MONOCYTE FUNCTION IN PATIENTS WITH BREAST CANCER STUARTA.TAYLOR, F.R.C.S.,t§ MYRTLE Y.GoRDoN,P~.D.,S GRAHAM A. CURRIE,M.D.,M.R.C.P.~ and VALERIE B. SHEPHERDS: Institute of Cancer Research, Sutton, Surrey, England We have measured peripheral blood monocyte function in breast cancer patients, using a maturation assay and a colony-forming assay. The results indicate that the changes in these two functions induced by local radiotherapy, are inversely related, so that patients showing an increase in circulating colony forming cells have monocytes with a decreased maturation index. Monocytes,
Colony forming units-C (CFU-C),
Breast cancer, Radiotherapy.
McCredie er ~1.‘~ concluded that B cells were de& cient in irradiated patients and that T cells, thought at the time to be important for immune surveillance, were normal or increased. However, there are no data to equate the numbers of cells forming E-rosettes with sheep red cells with the existence of an immune-suppressed state.’ It has been observed by Raila et al. I4 that successful radiotherapy is associated with improved immune status as measured by increased percentage blast transformation of lymphocytes and the increase of circulating T cells after treatment; the converse also appears to be true. The cause of the reduction of cell numbers or alteration of cell function is assumed to be the exposure of peripheral blood lymphocytes during irradiation. Similarly during the two minutes it takes for a mediastinal exposure, a considerable volume of blood passes through the irradiated field; this exposes the circulating cells of the mononuclear phagocyte system (MPS) to irradiation. It is known from animal data that the mature macrophage is relatively radioresistant. l2 However, the macrophage precursors are radiosensitive and the progeny of irradiated precursor cells show alterations in their development and function. In the irradiated mouse, the
INTRODUCTION The use of postoperative radiotherapy in breast cancer is well established; it has led to a reduction in the rate of local recurrence.6 It has been claimed that radiotherapy can have an adverse effect on survival in all stages of the disease,16 but recent observations on the early survival of patients with primary breast cancer have shown no such effect,17 and there is evidence to suggest that survival may be increased.g It has been suggested that radiation therapy could have an adverse effect by virtue of an immunosuppressive action on radiosensitive cells in the bone marrow and peripheral blood, and there is abundant evidence to show that a lymphopenia follows radiotherapy and can persist for up to 24 months after treatment has ended. However, as Alexander’ has pointed out, this cannot be equated with immunosuppression since the lymphopenia induced by radiotherapy may be as great as that seen in kidney transplant patients. Carcinoma patients treated by radiotherapy are not noticeably susceptible to infection, nor is infection a common cause of death as it is in recipients of kidney grafts or patients on high dose chemotherapy. The use of skin tests for sensitization have revealed widely divergent results14 and have led investigators to use in vitro tests of immune function, such as identification of T and B lymphocytes.
macrophage and granulocyte progenitors produce enlarged cells; irradiation also can reduce the effect of Corynebacterium Parvum on monocyte function in animals bearing tumors.7Js
tDepartment of Immunology. SDepartment of Biophysics. *Present address: King’s College Hospital, Denmark Hill, London SE5 9RS, England. Acknowledgements-The work was supported by a grant
from the Medical Research Council. Valerie B. Shepherd is supported by the Bud Flanagan Fund of the Royal Marsden Hospital. Reprint requests to: S.A. Taylor. Accepted for publication 21 August 1979. 2063
Radiation Oncology 0 Biology 0 Physics
2064
Although Stjernswtid l6 found no evidence of a monocytosis in post-mastectomy patients during ira consistent radiation, Rotman et a1.15 reported monocytosis in patients who were undergoing radiotherapy involving the chest or pelvis. In the present study, tests of monocyte function involving the maturation of peripheral blood monocytes,4 and the colony-forming ability of peripheral blood macrophage progenitor cells have been used to investigate the effects of X-irradiation on the MPS in women who are receiving post-mastectomy radiotherapy. METHODS AND MATERIALS Patients
Ten patients were studied; five had medial tumors and five had lateral tumors and positive axillary nodes. They received fractionated radiotherapy over a period of 6 weeks to a total dose of 4,800-5,000 rad. Blood samples were taken before radiotherapy, one and three weeks after the start of treatment and at the etid of the course of irradiation (6 weeks). A further sample was taken four weeks after the end of treatment (10 weeks) as a pilot study showed that monocyte maturation following irradiation had returned to approximate pre-treatment levels by this time. The standard five field Fletcher technique was used in all cases. This encompassed the homolateral chest wall, the internal mammary nodes, the homolateral axillary nodes and supraclavicular nodes. METHODS Defibrinated venous blood was centrifuged* as described by Boyum.2 Mononuclear cells were obtained from the interface, washed twice and resuspended in culture medium.? The Student “t” test was used in all cases. Colony-forming
assay
Colony-forming capacity was assayed in vitro in soft agar using peripheral blood leukocyte feeder layers as a source of colony-stimulating activity (CSA).13 One million mononuclear cells were plated as an upper agar layer with the additon of 2.5% rat hemolysate.8 The plates were incubated at 37°C in a gassed (10% COz in air) humidified atmosphere for 10 days. Those colonies which contained more than 50 cells and which demonstrated a definite central ‘cluster’ of cells to distinguish them from colony formation by cell migration, were counted by un*Ficoll-triosil (Lymphoprep.Nygaard). tRF’M1 1640 (plus 25mM Hepes and antibiotics).
November-December
1979, Vol. 5, NO. 11 and NO. 12
biased observers who had no knowledge of the identity of individual patients. Preparation of individual colonies$ showed characteristic monocytoid morphology and sample colonies (one taken from a plate at random after counting in each case) stained for non-specific esterase (NSE) were strongly positive. The results were expressed as the number of colony forming cells per lo6 mononuclear cells. Monocyte
maturation
assay
The mononuclear cell suspension obtained as described above was made up to 2 x lo6 cells per ml,t containing 50% fresh autologous serum. This was added in 100 ~1 volumes to the wells of microplates§ and incubated at 37°C in 2.5% CO, at 100% humidity for 7 days. One drop of the final cell suspension was added to each of 3 micro-slides and allowed to dry at room temperature. After fixation in formal acetone the slides were stained for non-specific esterase (a stain specific for monocytes) using the method described by Yam et al.lg After 7 days incubation the microplate wells were washed free of unattached cells and debris. Then 50 ~1 of 0.1 M citric acid solution containing 1:2000 crystal violet was added to each well and the plate incubated at 37°C for 10 minutes. After vigorous agitation the detached nuclei were counted in a hemocytometer. At least 5 replicate wells were counted in each case. Microscopic examination of the plates indicated that the agitation method removed all the cell nuclei from the plastic surface. The results were expressed as percentage maturation obtained by comparing the yield of attached macrophages as a percentage of the number of monocytes added using NSE positivity as a monocyte marker. The cell nuclei were readily identifiable as being reniform, whereas by contrast, contaminant lymphocytes in the culture (usually less than 1% after 3 washes of the wells) were readily distinguishable by their small nuclei and staining. There was no evidence that mitosis or colony formation took place in the wells during culture; because the day 7 macrophages appeared to be approximately 8 times (by area) larger than day 0 monocytes in culture, and were dendritic, the assay was judged to be one of maturation and differentiation as well as adherence and survival. RESULTS The results can be divided into two groups, depending upon the effects of X-irradiation on the monocyte functions tested. It was clear that when *May-Grunwald Giemsa stain. 03040 microplates. Falcon Plastics.
Irradiation and monocytes in breast cancer 0 S.A. TAYLOR et al.
the maturation of peripheral blood monocytes was depressed, the number of colony-forming cells rose and when maturation increased the colony-forming cells remained at the pre-treatment levels. Monocyte maturation: In eight patients, the maturation of peripheral blood monocytes was significantly depressed one week from the start of treatment (18.4 & 11.1%) compared with the pretreatment value of 41.6 * 26.2% @<0.05). As can be seen in Fig. 1, this depression persisted for the duration of treatment so that at three weeks it was 8.6 & 5.7% (pO.l). Two patients had elevated monocyte maturation during irradiation which fell to pre-treatment levels by one month after the end of treatment. Colony-forming ability: The same eight patients who had depressed monocyte maturation during radiotherapy showed a significant increase in colony-forming ability during irradiation (Fig. 2). At three weeks, the value was 13.8 -t 9.9 colonies/106 cells compared with 3.3 ? 2.4 colonies/106 cells before treatment (pcO.02). There was no difference between post-treatment levels (5.5 2 4.3 colonies/106 cells at 10 weeks) and pre-treatment levels @>O.l). The two patients who had elevated maturation of monocytes during irradiation showed no change in colony-forming cells throughout the time of the investigation. These results in the same two patients were not included in statistical analysis because we felt that their results represented a quite different response of the peripheral blood monocytes to radiotherapy in that there was an increase in the maturation percentage of peripheral blood monocytes during treatment, which was accompanied by a complete lack of response by the macrophage progenitor cells as assessed by this in vitro assay of colony formation in soft agar.
DISCUSSION The effects of X-irradiation on the monocyte functions measured by the two methods used in this study appear to be related. However, the numbers of circulating monocyte precursors, measured by the maturation assay, far outnumber the numbers of colony-forming cells (macrophage progenitor cells). It is uncertain whether the X-irradiation is having different effects on at least two distinct subpopulations of cells or whether the apparent difference results from different plating efficiencies of the cells in the two assay systems.
weeks
after
2065
onset of radiotherapy
Fig. 1. Changes in the incidence of colony-forming cells in the peripheral blood of breast cancer patients during radiotherapy. 0
weeks
after
34
onset of radiotherapy
Fig. 2. Changes in the maturation of monocytes in the peripheral blood of breast cancer patients during radiotherapy.
2066
Radiation Oncology 0 Biology 0 Physics
The mechanisms whereby irradiation may induce a monocytosis15 or its effects on peripheral blood monocytes are unknown. The studies of Dienstman and Defendi suggest that macrophages can be triggered into proliferation in two stages, an initial activation and a subsequent cell surface event. They make the assumption that full activation of monocytes occurs in vivo which could be brought about by a pathological process such as the growth of a tumor. Following activation, all that is required is a further non-specific stimulus for proliferation of cells to occur in vitro. This could be the mode of action of irradiation on macrophage progenitor cells where the signal for proliferation is the stimulus of treatment by irradiation and is entirely non-specific. This also might explain the rapid reversal of the effects of irradiation once the stimulus is removed so that the cells can then follow their ususal pattern of behavior. Actively phagocytic mature peritoneal macrophages from animals are relatively radioresistant and their phagocytic function continues when the cells are removed, after a dose of radiation, for functional assessment.” In contrast, the effect of irradiation on mouse macrophage progenitor cells is to reduce the phagocytic index and the in vitro cytotoxicity and anti-tumor effect of Corynebacterium Parvum. l8 Gadeberg et a1.7 showed that whole-body irradiation in mice reduced the phagocytic activity of mouse peritoneal macrophages and concluded that because irradiation is antiproliferative3 it could inhibit the formation of precursor cells in the bone marrow, thus reducing the number of monocytes in the blood available for maturation into macrophages. Equally, these effects could have been on precursors in the blood or even in the peritoneal cell population which always con-
November-December 1979, Vol. 5, No. 11 and No. 12
tains a proportion of cells which have recently arrived from the circulation. In the present series of patients where maturation was decreased, the defect did not appear to be a simple numerical one since there were ample NSE positive cells in the smear i.e., there was no monocytopenia. Indeed, it has been shown that there is a monocytosis during radiotherapy.‘” The primary cause of the effects on monocyte function described here must be initiated in the field of irradiation. The irradiated tissues include the site of the tumor and the peripheral blood cells which circulate through the irradiated volume during exposure but they include only a very small proportion of the total bone marrow. This contrasts with the situation when small laboratory animals are irradiated; whole body doses are usually given resulting in the destruction of a high proportion of macrophage progenitor cells.” It follows that quite a different mechanism is at work in the case of local irradiation. This may operate by creating cell debris at the site of irradiation with subsequent macrophage migration and stimulation of the marrow, to release more (less mature?) progenitor cells into the circulation; this results in increased numbers of colony-forming cells and a lower maturation index. Alternatively, the mechanism may be more direct and result from the destruction of cells circulating through the irradiated field during exposure. Whether monocyte function is suppressed or stimulated, the effects are short-lived and cannot be used to support the concept that radiotherapy has deleterious effects which are mediated through the monocyte/macrophage system.
REFERENCES 1. Alexander, P.: The bogey of the immunosuppressive action of local radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 1: 369-371, 1976. of mononuclear cells and 2. Boyum, A.: Isolation granulocytes from human blood. &and. J. Clin. Lab. Invest. 21: 77-89, 1968. 3. Calabresi, P., Parks, R.E.: Chemotherapy of neoplastic diseases. In The Pharmacological Basis of Therapeatics, eds. Goodman, L.S. and Gilman, A. The Macmillan Company. London and Toronto. 1970, pp. 13441396. 4. Currie, G.A., Hedley, D.W.: Monocytes and macrophages in malignant melanoma. I. Peripheral blood macrophage precursors. Brit. J. Cancer 36: l-6, 1977. 5. Dienstman, S.R., Defendi, J.: Necessary and sufficient conditions for recruitment of macrophages into the cell cycle. Exp. Cell. Res. 115: 191-199, 1978. 6. Fisher, B., Slack, N.H., Cavanaugh, P.J., Gardner, B., Ravdin, R.G.: Postoperative radiotherapy in the treatment of breast cancer. Ann. Surg. 172: 711-732, 1970.
7. Gadeberg, O.V., Rhodes, J.M., Larsen, S.O.: The effeet of various immunosuppressive agents on mouse peritoneal macrophages and on the in vitro phagocytosis of Escherichia coli 04:K3:HS and degradation of lzs I-labelled HSA antibody complexes by these cells. Immunology 28: 5970, 1975. 8. Gordon, M.Y.: Circulating inhibitors of granulopoiesis in patients with aplastic anaemia. Brit. J. Haematol. 39: 491-496, 1978. 9. Host, H., Brennhovd, 1.0.: The effect of postoperative radiotherapy in breast cancer. Int. J. Rad. Oncol. Biol. Phys. 2: 1061-1067, 1977. 10. McCredie, J.A., Inch, W.R., Sutherland, R.M.: Effects of postoperative radiotherapy on peripheral blood lymphocytes in patients with carcinoma of the breast. Cancer 29: 349-356, 1972. 11. Metcalf, D., Johnson, G.R., Wilson, J.: Radiation induced enlargement of granulocytic and macrophage progenitor cells in mouse bone marrow. Exp. Haematol.
5: 299-309,
1977.
Irradiation and monocytes
in breast cancer 0 S.A. TAYLOR et~1.
12. Perkins,
E.H., Nettesheim, P., Morita, T.: Radioresistance of the engulfing and degradation capacities of peritoneal phagocytes to kiloroentgen X-ray doses. J. Reticuloendothelial Sot. 3: 71-82, 1966. 13. Pike, B.L., Robinson, W.A.: Human bone marrow colony growth in agar gel. J. Cell Physiol.76: 77-84, 1970. 14.Ratla, S.,
Yang, S.J., Meleka, F.: Changes in cell patients mediated immunity in undergoing radiotherapy. Cancer 41: 10761086, 1978. 15. Rotman, M., Ansley, H., Rogow, I., Stowe, S.: Monocytosis: A new observation during radiotherapy. Int. J. Rad. Oncol. Biol. Phys. 2: 117-121, 1977.
2067
16. Stjemsward, J.: Decreased survival related to irradiation post-operatively in early operable breast cancer. Lancet ii, No. 7892, 1285-1286, Nov. 30, 1974. 17. Turnbull, A.R., Chant, A.D.B., Buchanan, R.B., Turner, D.T.L., Shepherd, J.M., Fraser, J.D.: Treatment of early breast cancer. Lancet ii, 7-9, 1978. Ghaffer, A., Whitehead, V.A.: 18. Woodruff, M.F.A., Modification of the effect of C. parvum on macrophage activity and tumour growth by X-irradiation. Int. J. Cancer 17: 6.52-658, 1977. 19. Yam, L.T., Li, C.Y., Crosby, W.H.: Cytochemical Am. J. identification of monocytes and granulocytes. Clin. Path. 55: 283-290, 1971.
Int. .I. Radiation Oncology Biol. Phys., Vol. 5, pp. 2063-2067 o Pergamon Press Ltd., 1979. Printed in the U.S.A.
??Brief Communication
IRRADIATION AND MONOCYTE FUNCTION IN PATIENTS WITH BREAST CANCER STUARTA.TAYLOR, F.R.C.S.,t§ MYRTLE Y.GoRDoN,P~.D.,S GRAHAM A. CURRIE,M.D.,M.R.C.P.~ and VALERIE B. SHEPHERDS: Institute of Cancer Research, Sutton, Surrey, England We have measured peripheral blood monocyte function in breast cancer patients, using a maturation assay and a colony-forming assay. The results indicate that the changes in these two functions induced by local radiotherapy, are inversely related, so that patients showing an increase in circulating colony forming cells have monocytes with a decreased maturation index. Monocytes,
Colony forming units-C (CFU-C),
Breast cancer, Radiotherapy.
McCredie er ~1.‘~ concluded that B cells were de& cient in irradiated patients and that T cells, thought at the time to be important for immune surveillance, were normal or increased. However, there are no data to equate the numbers of cells forming E-rosettes with sheep red cells with the existence of an immune-suppressed state.’ It has been observed by Raila et al. I4 that successful radiotherapy is associated with improved immune status as measured by increased percentage blast transformation of lymphocytes and the increase of circulating T cells after treatment; the converse also appears to be true. The cause of the reduction of cell numbers or alteration of cell function is assumed to be the exposure of peripheral blood lymphocytes during irradiation. Similarly during the two minutes it takes for a mediastinal exposure, a considerable volume of blood passes through the irradiated field; this exposes the circulating cells of the mononuclear phagocyte system (MPS) to irradiation. It is known from animal data that the mature macrophage is relatively radioresistant. l2 However, the macrophage precursors are radiosensitive and the progeny of irradiated precursor cells show alterations in their development and function. In the irradiated mouse, the
INTRODUCTION The use of postoperative radiotherapy in breast cancer is well established; it has led to a reduction in the rate of local recurrence.6 It has been claimed that radiotherapy can have an adverse effect on survival in all stages of the disease,16 but recent observations on the early survival of patients with primary breast cancer have shown no such effect,17 and there is evidence to suggest that survival may be increased.g It has been suggested that radiation therapy could have an adverse effect by virtue of an immunosuppressive action on radiosensitive cells in the bone marrow and peripheral blood, and there is abundant evidence to show that a lymphopenia follows radiotherapy and can persist for up to 24 months after treatment has ended. However, as Alexander’ has pointed out, this cannot be equated with immunosuppression since the lymphopenia induced by radiotherapy may be as great as that seen in kidney transplant patients. Carcinoma patients treated by radiotherapy are not noticeably susceptible to infection, nor is infection a common cause of death as it is in recipients of kidney grafts or patients on high dose chemotherapy. The use of skin tests for sensitization have revealed widely divergent results14 and have led investigators to use in vitro tests of immune function, such as identification of T and B lymphocytes.
macrophage and granulocyte progenitors produce enlarged cells; irradiation also can reduce the effect of Corynebacterium Parvum on monocyte function in animals bearing tumors.7Js
tDepartment of Immunology. SDepartment of Biophysics. *Present address: King’s College Hospital, Denmark Hill, London SE5 9RS, England. Acknowledgements-The work was supported by a grant
from the Medical Research Council. Valerie B. Shepherd is supported by the Bud Flanagan Fund of the Royal Marsden Hospital. Reprint requests to: S.A. Taylor. Accepted for publication 21 August 1979. 2063
Radiation Oncology 0 Biology 0 Physics
2064
Although Stjernswtid l6 found no evidence of a monocytosis in post-mastectomy patients during ira consistent radiation, Rotman et a1.15 reported monocytosis in patients who were undergoing radiotherapy involving the chest or pelvis. In the present study, tests of monocyte function involving the maturation of peripheral blood monocytes,4 and the colony-forming ability of peripheral blood macrophage progenitor cells have been used to investigate the effects of X-irradiation on the MPS in women who are receiving post-mastectomy radiotherapy. METHODS AND MATERIALS Patients
Ten patients were studied; five had medial tumors and five had lateral tumors and positive axillary nodes. They received fractionated radiotherapy over a period of 6 weeks to a total dose of 4,800-5,000 rad. Blood samples were taken before radiotherapy, one and three weeks after the start of treatment and at the etid of the course of irradiation (6 weeks). A further sample was taken four weeks after the end of treatment (10 weeks) as a pilot study showed that monocyte maturation following irradiation had returned to approximate pre-treatment levels by this time. The standard five field Fletcher technique was used in all cases. This encompassed the homolateral chest wall, the internal mammary nodes, the homolateral axillary nodes and supraclavicular nodes. METHODS Defibrinated venous blood was centrifuged* as described by Boyum.2 Mononuclear cells were obtained from the interface, washed twice and resuspended in culture medium.? The Student “t” test was used in all cases. Colony-forming
assay
Colony-forming capacity was assayed in vitro in soft agar using peripheral blood leukocyte feeder layers as a source of colony-stimulating activity (CSA).13 One million mononuclear cells were plated as an upper agar layer with the additon of 2.5% rat hemolysate.8 The plates were incubated at 37°C in a gassed (10% COz in air) humidified atmosphere for 10 days. Those colonies which contained more than 50 cells and which demonstrated a definite central ‘cluster’ of cells to distinguish them from colony formation by cell migration, were counted by un*Ficoll-triosil (Lymphoprep.Nygaard). tRF’M1 1640 (plus 25mM Hepes and antibiotics).
November-December
1979, Vol. 5, NO. 11 and NO. 12
biased observers who had no knowledge of the identity of individual patients. Preparation of individual colonies$ showed characteristic monocytoid morphology and sample colonies (one taken from a plate at random after counting in each case) stained for non-specific esterase (NSE) were strongly positive. The results were expressed as the number of colony forming cells per lo6 mononuclear cells. Monocyte
maturation
assay
The mononuclear cell suspension obtained as described above was made up to 2 x lo6 cells per ml,t containing 50% fresh autologous serum. This was added in 100 ~1 volumes to the wells of microplates§ and incubated at 37°C in 2.5% CO, at 100% humidity for 7 days. One drop of the final cell suspension was added to each of 3 micro-slides and allowed to dry at room temperature. After fixation in formal acetone the slides were stained for non-specific esterase (a stain specific for monocytes) using the method described by Yam et al.lg After 7 days incubation the microplate wells were washed free of unattached cells and debris. Then 50 ~1 of 0.1 M citric acid solution containing 1:2000 crystal violet was added to each well and the plate incubated at 37°C for 10 minutes. After vigorous agitation the detached nuclei were counted in a hemocytometer. At least 5 replicate wells were counted in each case. Microscopic examination of the plates indicated that the agitation method removed all the cell nuclei from the plastic surface. The results were expressed as percentage maturation obtained by comparing the yield of attached macrophages as a percentage of the number of monocytes added using NSE positivity as a monocyte marker. The cell nuclei were readily identifiable as being reniform, whereas by contrast, contaminant lymphocytes in the culture (usually less than 1% after 3 washes of the wells) were readily distinguishable by their small nuclei and staining. There was no evidence that mitosis or colony formation took place in the wells during culture; because the day 7 macrophages appeared to be approximately 8 times (by area) larger than day 0 monocytes in culture, and were dendritic, the assay was judged to be one of maturation and differentiation as well as adherence and survival. RESULTS The results can be divided into two groups, depending upon the effects of X-irradiation on the monocyte functions tested. It was clear that when *May-Grunwald Giemsa stain. 03040 microplates. Falcon Plastics.
Irradiation and monocytes in breast cancer 0 S.A. TAYLOR et al.
the maturation of peripheral blood monocytes was depressed, the number of colony-forming cells rose and when maturation increased the colony-forming cells remained at the pre-treatment levels. Monocyte maturation: In eight patients, the maturation of peripheral blood monocytes was significantly depressed one week from the start of treatment (18.4 & 11.1%) compared with the pretreatment value of 41.6 * 26.2% @<0.05). As can be seen in Fig. 1, this depression persisted for the duration of treatment so that at three weeks it was 8.6 & 5.7% (p
DISCUSSION The effects of X-irradiation on the monocyte functions measured by the two methods used in this study appear to be related. However, the numbers of circulating monocyte precursors, measured by the maturation assay, far outnumber the numbers of colony-forming cells (macrophage progenitor cells). It is uncertain whether the X-irradiation is having different effects on at least two distinct subpopulations of cells or whether the apparent difference results from different plating efficiencies of the cells in the two assay systems.
weeks
after
2065
onset of radiotherapy
Fig. 1. Changes in the incidence of colony-forming cells in the peripheral blood of breast cancer patients during radiotherapy. 0
weeks
after
34
onset of radiotherapy
Fig. 2. Changes in the maturation of monocytes in the peripheral blood of breast cancer patients during radiotherapy.
2066
Radiation Oncology 0 Biology 0 Physics
The mechanisms whereby irradiation may induce a monocytosis15 or its effects on peripheral blood monocytes are unknown. The studies of Dienstman and Defendi suggest that macrophages can be triggered into proliferation in two stages, an initial activation and a subsequent cell surface event. They make the assumption that full activation of monocytes occurs in vivo which could be brought about by a pathological process such as the growth of a tumor. Following activation, all that is required is a further non-specific stimulus for proliferation of cells to occur in vitro. This could be the mode of action of irradiation on macrophage progenitor cells where the signal for proliferation is the stimulus of treatment by irradiation and is entirely non-specific. This also might explain the rapid reversal of the effects of irradiation once the stimulus is removed so that the cells can then follow their ususal pattern of behavior. Actively phagocytic mature peritoneal macrophages from animals are relatively radioresistant and their phagocytic function continues when the cells are removed, after a dose of radiation, for functional assessment.” In contrast, the effect of irradiation on mouse macrophage progenitor cells is to reduce the phagocytic index and the in vitro cytotoxicity and anti-tumor effect of Corynebacterium Parvum. l8 Gadeberg et a1.7 showed that whole-body irradiation in mice reduced the phagocytic activity of mouse peritoneal macrophages and concluded that because irradiation is antiproliferative3 it could inhibit the formation of precursor cells in the bone marrow, thus reducing the number of monocytes in the blood available for maturation into macrophages. Equally, these effects could have been on precursors in the blood or even in the peritoneal cell population which always con-
November-December 1979, Vol. 5, No. 11 and No. 12
tains a proportion of cells which have recently arrived from the circulation. In the present series of patients where maturation was decreased, the defect did not appear to be a simple numerical one since there were ample NSE positive cells in the smear i.e., there was no monocytopenia. Indeed, it has been shown that there is a monocytosis during radiotherapy.‘” The primary cause of the effects on monocyte function described here must be initiated in the field of irradiation. The irradiated tissues include the site of the tumor and the peripheral blood cells which circulate through the irradiated volume during exposure but they include only a very small proportion of the total bone marrow. This contrasts with the situation when small laboratory animals are irradiated; whole body doses are usually given resulting in the destruction of a high proportion of macrophage progenitor cells.” It follows that quite a different mechanism is at work in the case of local irradiation. This may operate by creating cell debris at the site of irradiation with subsequent macrophage migration and stimulation of the marrow, to release more (less mature?) progenitor cells into the circulation; this results in increased numbers of colony-forming cells and a lower maturation index. Alternatively, the mechanism may be more direct and result from the destruction of cells circulating through the irradiated field during exposure. Whether monocyte function is suppressed or stimulated, the effects are short-lived and cannot be used to support the concept that radiotherapy has deleterious effects which are mediated through the monocyte/macrophage system.
REFERENCES 1. Alexander, P.: The bogey of the immunosuppressive action of local radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 1: 369-371, 1976. of mononuclear cells and 2. Boyum, A.: Isolation granulocytes from human blood. &and. J. Clin. Lab. Invest. 21: 77-89, 1968. 3. Calabresi, P., Parks, R.E.: Chemotherapy of neoplastic diseases. In The Pharmacological Basis of Therapeatics, eds. Goodman, L.S. and Gilman, A. The Macmillan Company. London and Toronto. 1970, pp. 13441396. 4. Currie, G.A., Hedley, D.W.: Monocytes and macrophages in malignant melanoma. I. Peripheral blood macrophage precursors. Brit. J. Cancer 36: l-6, 1977. 5. Dienstman, S.R., Defendi, J.: Necessary and sufficient conditions for recruitment of macrophages into the cell cycle. Exp. Cell. Res. 115: 191-199, 1978. 6. Fisher, B., Slack, N.H., Cavanaugh, P.J., Gardner, B., Ravdin, R.G.: Postoperative radiotherapy in the treatment of breast cancer. Ann. Surg. 172: 711-732, 1970.
7. Gadeberg, O.V., Rhodes, J.M., Larsen, S.O.: The effeet of various immunosuppressive agents on mouse peritoneal macrophages and on the in vitro phagocytosis of Escherichia coli 04:K3:HS and degradation of lzs I-labelled HSA antibody complexes by these cells. Immunology 28: 5970, 1975. 8. Gordon, M.Y.: Circulating inhibitors of granulopoiesis in patients with aplastic anaemia. Brit. J. Haematol. 39: 491-496, 1978. 9. Host, H., Brennhovd, 1.0.: The effect of postoperative radiotherapy in breast cancer. Int. J. Rad. Oncol. Biol. Phys. 2: 1061-1067, 1977. 10. McCredie, J.A., Inch, W.R., Sutherland, R.M.: Effects of postoperative radiotherapy on peripheral blood lymphocytes in patients with carcinoma of the breast. Cancer 29: 349-356, 1972. 11. Metcalf, D., Johnson, G.R., Wilson, J.: Radiation induced enlargement of granulocytic and macrophage progenitor cells in mouse bone marrow. Exp. Haematol.
5: 299-309,
1977.
Irradiation and monocytes
in breast cancer 0 S.A. TAYLOR et~1.
12. Perkins,
E.H., Nettesheim, P., Morita, T.: Radioresistance of the engulfing and degradation capacities of peritoneal phagocytes to kiloroentgen X-ray doses. J. Reticuloendothelial Sot. 3: 71-82, 1966. 13. Pike, B.L., Robinson, W.A.: Human bone marrow colony growth in agar gel. J. Cell Physiol.76: 77-84, 1970. 14.Ratla, S.,
Yang, S.J., Meleka, F.: Changes in cell patients mediated immunity in undergoing radiotherapy. Cancer 41: 10761086, 1978. 15. Rotman, M., Ansley, H., Rogow, I., Stowe, S.: Monocytosis: A new observation during radiotherapy. Int. J. Rad. Oncol. Biol. Phys. 2: 117-121, 1977.
2067
16. Stjemsward, J.: Decreased survival related to irradiation post-operatively in early operable breast cancer. Lancet ii, No. 7892, 1285-1286, Nov. 30, 1974. 17. Turnbull, A.R., Chant, A.D.B., Buchanan, R.B., Turner, D.T.L., Shepherd, J.M., Fraser, J.D.: Treatment of early breast cancer. Lancet ii, 7-9, 1978. Ghaffer, A., Whitehead, V.A.: 18. Woodruff, M.F.A., Modification of the effect of C. parvum on macrophage activity and tumour growth by X-irradiation. Int. J. Cancer 17: 6.52-658, 1977. 19. Yam, L.T., Li, C.Y., Crosby, W.H.: Cytochemical Am. J. identification of monocytes and granulocytes. Clin. Path. 55: 283-290, 1971.