Immunobiol., vol. 181, pp. 288-297 (1990)
Department of Hematology, Academy of Medicine, Poznan, Poland
Impaired Release of Colony Stimulating Activity by Monocytes from Hodgkin's Disease in Response to Phorbol Myristate Acetate Activation JANUSZ HANSZ, KRZYSZTOF SAWIN-SKI, and TOMASZ WOZNY
Received July 10, 1989· Accepted in Revised Form May 21,1990
Abstract We assessed the humoral effect of resting and phorbol esters preincubated monocytes from Hodgkin's disease patients (HDMo) and healthy subjects (nMo), on granulocyte progenitors (CFU-dG) growth using a double diffusion chamber technique. The release of colony stimulating activity and indomethacin-dependent inhibitors by resting HDMo and nMo was found to be cell-concentration dependent. However, phorbol myristate acetate preincubated HDMo (PMA-HDMo) in contrast to nMo at low concentrations (2.5 x 104 ) were unable to increase the CFU-dG growth stimulation. On the other hand, at a higher cell number (5 x 104 ), phorbol treated HDMo stimulated the myeloid colony formation, whereas nMo suppressed the CFU-dG proliferation. Further enhancement of HDMo and nMo concentrations induced a pronounced inhibition of CFU-dG-derived colony formation, caused by an increased PGE 2 production. After incubation with the cyclooxygenase inhibitor-indomethacin, PMA-HDMo showed considerably more granulocyte colony formation than nMo. Our results suggest that the observed abnormalities in the function of HDMo could be associated with an excessive production of PGE 2 and a general dysfunction of these cells in Hodgkin's disease.
Introduction Monocytes (Mo) playa crucial role in the regulation of hematopoiesis, at least in culture. Directly and in cooperation with other cells, they produce humoral factors that stimulate or suppress the hematopoietic precursor proliferation (1). Monocyte derived colony stimulating factors (CSF), like other CSFs, are glycoproteins essential for myeloid colony formation (2). Monocytes also release potent inhibitors of granulocyte progenitor growth, such as prostaglandin E (3), acidic isoferritins (4), and tumor necrosis factor
(TNF) (5). Abbreviations: CFU-dG = colony forming unit-granulocyte (diffusion chamber); CSA = colony stimulating activity; FCS = fetal calf serum; HBSS = Hanks' balanced salt solution; HD = Hodgkin's disease; Mo = monocytes; NBT = nitro blue tetrazolium; PBS = phosphate-buffered saline; PMA = phorbol12-myristate 13-acetate; TNF = tumor necrosis factor; PGE2 = prostaglandin E2 .
CSA release by Hodgkin's disease monocytes . 289
In Hodgkin's disease (HD) the monocytes reveal many abnormalities, e.g. reduced phagocytic activity (6), decreased chemotactic response (7), diminished expression of class II MHC antigens (8) and an increased prostaglandin production (9, 10). These abnormalities may be of great significance for the impaired immunity in HD. Little is known, however, whether HD monocytes demonstrate a normal or defective function in the regulation of hematopoiesis. In the present study, we were investigating the effects of resting and PMA-activated HDMo on granulocyte progenitor growth and subsequently found that these cells, being already preactivated, revealed an altered response to phorbol stimulation and an abnormal modulation of myeloid colony formation in diffusion chamber culture. Materials and Methods Patients Peripheral blood (PB) and bone marrow (BM) samples were collected from 15 patients with HD in the clinical/pathological stage III or IV. The specimens for control experiments were obtained from 15 hematologically normal individuals undergoing cardiac surgery. Both PB and BM samples were gathered after informed consent had been obtained from the patients in accordance with the guidlines of the Academy of Medicine Committee, for protection of human subjects. Cell separation Bone marrow mononuclear cells isolated by Ficoll/Hypaque (specific gravity 1.077; Pharmacia, Uppsala, Sweden) density gradient centrifuging at 400 g for 15 min, after washing three times, were then resuspended in McCoy's medium (Flow, U.K.) containing 20 % fetal calf serum (FCS, Calbiochem, Switzerland), 200 U/ml penicillin G and 200 U/ml streptomycin. The viability of cells, assessed by Trypan Blue exclusion, exceeded 96 %. Monocytes were separated from PB. The blood was diluted in an equal volume of Hanks' Balanced Salt Solution (HBSS, Biomed, Poland) and the mononuclear cells were isolated by Ficoll/Hypaque density gradient centrifuging at 400 g for 40 min. Cells from the interphase, after washing three times and resuspending in McCoy'S medium supplemented with 20 % FCS in a final concentration of 106 cells/ml, were incubated in plastic Petri dishes at 37°C in 5 % CO 2 • After 1.5 h, the non-adherent cells were removed by gently washing the dishes with HBSS. The adherent cells were recovered by scraping and then suspended in McCoy's medium enriched with FCS and antibiotics in the same concentrations as above. The adherent cell suspension consisted of monocytes (mean 76 %), neutrophils (20 %), eosinophils (2 %), and lymphocytes (2 %) as determined by the May-Grunwald-Giemsa method and by the nonspecific esterase staining. In some experiments, monocytes were incubated for 5 min, at 37°C with phorbol 12myristate 13-acetate (Sigma, USA) in a final concentration of 1 flg/ml of McCoy's medium containing 106 cells/m!. After incubation, monocytes were washed twice and resuspended in McCoy's medium supplemented with 20 % FCS and antibiotics. In the control experiments, the cells were incubated under the same conditions, except without phorbol myristate acetate (PMA). The viability of monocytes each time exceeded 90 %. Nitroblue tetrazolium (NBT) reduction test The NBT reduction test was performed following methods used by PARK et al. (11). Monocyte suspension (5 x 105 /ml PBS) was mixed with an equal volume of 0.2 % nitro blue
290 . ]. HANSZ, K. SAWINSKI, and T. WOZNY Table 1. NBTa reduction test of monocytes from normal subjects and patients with Hodgkin's disease Material
Monocytes resting
PMA-treated b
Healthy subjects
22 ± Se
61 ± 7
Hodgkin's disease patients
4S ± 10
57 ± 11
a NBT - nitro blue tetrazolium monocytes incubated with phorbol12-myristate 13-acetate (2 !-Ig/m1/106 cells, 5 min., 37°C) C percent of NBT-positive cells (mean ± standard deviation of six separate experiments)
b
tetrazolium (NBT, Sigma, USA) in 0.15 M NaCI. The cells were incubated for 30 min at 37°C and then again for 30 min at IS dc. The percentage of formazan containing cells was determined on glass slides stained by the Pappenheim method.
Superoxide anion (Oi) production The O 2 production was estimated by the method described by COHEN and CHOVANIEC (12). Briefly, 0.1 ml of monocyte suspension (5 x 105 cells/ml PBS) was mixed with 0.3 ml of cytochrom C solution (2 mg/ml; WSiSz, Krakow, Poland). After incubation (15 min, 3rq superoxide dismutase (2 mg/ml, Sigma, USA) was added. The extinction of supernatant was measured at 550 nm wave length. The control sample was prepared in the same manner but without monocytes. 0;; production was calculated in nmol of reduced cytochrom C for 106 monocytes.
Diffusion chamber culture The agar diffusion chamber technique used for granulocyte colony formation was a modification of the method of GORDON and BLACKETT (13). Briefly, the diffusion chambers (DC) used in this study, consisted of two 400 !-II compartments separated by a membrane filter (Sartorius, pore size 0.45 !-1m). The chambers were covered on one side by a membrane filter and sealed on the other by a glass slide. Bone marrow cells (2 x 105/0.4 ml), resuspended in McCoy's medium with FCS and antibiotics in 0.3 % agar (Difco, USA), were introduced into the DC compartment covered on both sides by a filter. Monocytes placed in the second DC-compartment were suspended in the same medium, but with 0.5 % agar. In some of the experiments, together with monocytes, indomethacin (Merck, FRG) was incorporated into the chambers at a final concentration of Table 2. Superoxide anion production by resting and phorbol myristate acetate preincubated normal and Hodgkin's disease monocytes Material
Monocytes resting
Healthy subjects Hodgkin's disease patients
6.6 ± 2.Sb 15.2 ± 5.7
PMA-treateda 19.9±5.5 17.l±7.2
a monocytes incubated with phorbol12-myristate 13-acetate (2 !-Ig/m1/106 cells, 5 min., 37°C) 6 b nmol superoxide anions!10 cells (mean ± standard deviation of six separate experiments)
CSA release by Hodgkin's disease monocytes . 291 1.7 mmolli. After gelling of agar at room temperature, the DCs were implanted into the peritoneal cavity of rats (female, Wi star strain, weighing 200-300 g) which had been irradiated with X-ray (6.0 Gy) 2 h prior to implantation. The cultures were performed in quadruplicate for each set of experiments. On day 9 the number of colonies (> 20 cells) was determined. The cellular composition of colonies was determined on slides stained by the May-Griinwald-Giemsa method and also evaluated for myeloperoxidase, naphthyl-ASD chloroacetate esterase and acid a-naphthylacetate esterase. The colonies consisted of myeloblasts (20 %), myelocytes (39 %), metamyelocytes (25 %) and mature granulocytes (16 %). No significant difference between the cellular composition of colonies grown from normal and HD marrow was seen. Results were analysed using Student's t test. Differences were considered statistically significant if p < 0.05.
Results
NBT reduction test and superoxide anion production in resting and PMApreincubated normal (nMo) and Hodgkin's disease monocytes (HDMo)
In comparison with resting nMo, after incubation with PMA, an increased number of cells contained formazan and revealed an augmented superoxide anion production (p < 0.05; Table 1). In contrast to nMo, resting HDMo already displayed an enhanced superoxide production and an increased formazan content (p < 0.05). Preincubation with PMA did not change these characteristics (p > 0.05; Table 2).
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MONOCYTES Figure 1. Effect of normal monocytes (Mo) on autologous marrow granulocytic precursors proliferation in double diffusion chamber culture. Resting Mo (e--e), Mo incubated with 1 f.lg/ml phorbol12-myristate 13-acetate (PMA-Mo) (0---0), PMA-Mo and 1.7 mmolll indomethacin (0---0). Each point represents a mean ± standard deviation from four experiments done in quadruplicate. The results are expressed as a percentage of control (colony formation without monocytes).
292 .
J. HANSZ, K. SAWINSKI, and T. WOZNY
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Figure 2. Effect of monocytes from Hodgkin's disease patients (HDMo) on autologous marrow granulocytic precursors proliferation in diffusion chamber culture. Resting HDMo (e--e), HDMo incubated with 1 flg/ml phorbol12-myristate 13-acetate (PMA-HDMo) (0---0), PMA-HDMo and, 1.7 mmol!l indomethacin (0---0). Each point represents a mean ± standard deviation from four experiments done in quadruplicate. The results are expressed as a percentage of control (colony formation without monocytes).
Effect of normal monocytes (nMo) on autologous marrow granulocytic precursors prollferation In control cultures (without monocytes), the marrow granulocyte progenitors formed 27 ± 3 colonies (mean ± SD from four experiments done in quadruplicate). In experiments with monocytes, the myeloid colony formation was dependent on the number of monocytes introduced into the chambers (Fig. 1). The highest cloning efficiency was observed at 5 x 104 nMo and the colony number (50 ± 7) increased almost 85 % over the control value (p < 0.01). At higher Mo concentrations the colony count was diminished. In cultures with 1.0 x 105 nMo the myeloid colony formation was within the range of the control value (p > 0.05). The most significant stimulating effect of PMA-preincubated nMo was observed at a lower concentration of these cells (2.5 x 104 /chamber) in comparison with resting nMo. With higher Mo numbers the colony count was markedly lower (p < 0.05). The inhibition of CFU-dG growth at 5 x 10\ 7.5 X 10 4 and 1.0 x 105 nMo was entirely abolished when indomethacin, together with Mo suspension, was introduced into the chambers and the myeloid colony formation was within the range observed for maximum stimulation in experiments performed with resting monocytes (p> 0.05). Resting and activated nMo exerted a similar CFU-dG growth enhancing effect independently, whether normal or HD marrow cells were used (Fig. 3A).
CSA release by Hodgkin's disease monocytes . 293
Effect of Hodgkin's disease monocytes (HDMo) on autologous marrow granulocytic precursors proliferation In control experiments (without monocytes), the cloning efficiency of HD marrow cells was significantly lower compared with the normal controls (19 ± 3; P < 0.05). Resting HDMo promoted autologous CFU-dG growth like the monocytes from healthy subjects (Fig. 2). Also the greatest stimulating effect was detected at 5 x 104 Mo and the colony number (43 ± 6) exceeded the control value by greater than 90 % (p < 0.01). In contrast to the results obtained with PMA-treated nMo, however, PMA-preincubated HDMo, at a lower concentration (2.5 x 104 ), stimulated CFU-dG-derived colony formation to a much lesser degree (p < 0.05). The maximum increase in CFU-dG growth was noted at 5.0 x 104 Mo, similarly as in cultures with unstimulated cells (p> 0.05). Higher monocyte concentration, induced a suppression of the myeloid colony formation in comparison with the effect of resting cells. As in the case of nMo, in the presence of indomethacin, no inhibition of CFU-dG proliferation at higher HDMo concentrations was observed. It should be stressed, that under these experimental conditions, although the increase in cloning efficiency observed in investigations with HDMo was relatively higher than that noted in comparable cultures with nMo, the absolute number of colonies formed was similar (29 ± 5 and 44 ± 11 respectively; p > 0.05). There was no relationship between HDMo functional impairment and the clinical characteristics. However, it should be emphasized that only patients in advanced stages of the disease were studied.
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Figure 3. Effect of resting and phorbol myristate acetate (PMA) preincubated normal (A) and Hodgkin's disease (B) monocytes on allogenous marrow granulocytic precursors proliferation in diffusion chamber culture. (A): normal resting monocytes (e--e) or normal PMAtreated monocytes (0---0) + Hodgkin's disease marrow mononuclear cells. (B): Hodgkin's disease resting monocytes (e--e) or PMA-treated Hodgkin's disease monocytes (0---0) + normal marrow mononuclear cells.
294 .
J. HANSZ, K.
SAWINSKI,
and T.
WOZNY
The effect of resting and PMA-treated HDMo on the growth of marrow CFU-dG from healthy subjects was comparable to that observed in experiments with autologous marrow cells (Fig. 3B).
Discussion In this paper, we are presenting evidence that resting and phorbol treated monocytes from Hodgkin's disease patients and healthy subjects produce growth-promoting and inhibiting activities for granulocyte progenitors, which proliferate and differentiate in diffusion chamber culture. It should be emphasized, that this population of granulocyte progenitors differs essentially from that which gives rise to myeloid colonies in vitro (14, 15). The monocyte effect on autologous marrow granulocytic progenitor growth, both in Hodgkin's disease and in the control, was concentration dependent. Dose response curves indicated that lower Mo numbers induced an increase in cloning efficiency, whereas at higher concentrations a marked suppression of CFU-dG-derived colony formation was observed. These findings are not surprising since monocytes are a potent source of CSF, which is essential for granulocytic colony growth in vitro and in vivo in diffusion chamber culture (2, 16). The decline of the colony formation at higher Mo concentrations was clearly due to prostaglandin release from monocytes, since it was completely abolished under the influence of cyclooxygenase inhibitor-indomethacin (17). This creates an argument against the possible effect of other inhibitors, such as TNF or interferon, which could be released by monocytes and in a peritoneal environment (5, 18). No difference in granulocyte progenitors growth promoting effect of normal or HD monocytes was observed unless PMA-treated cells were tested. Phorbol esters, which are protein kinase C activators, became a useful tool in the study of signal transduction and cell activation (19, 20). The present work has confirmed the stimulating property of PMA on monocytes by demonstrating the changes in super oxide production and in NBT reduction test. Unfortunately, the monocyte suspension used in these experiments was to some degree contaminated by granulocytes and lymphocytes. It seems rather doubtful that these cells could mediate in the PMA activation of monocytes. However, unless a pure population of cells is used, such a possibility can not be ruled out. Of interest was the finding that intact HDMo, in contrast to normal cells, being already activated, were insensitive to further stimulation by PMA. This may indicate that HDMo are unable to respond to protein kinase C activators. The present study clearly shows that monocyte from healthy subjects, after incubation with PMA, revealed their maximum CFU-dG growth promoting effect at lower cell concentrations when compared with resting
CSA release by Hodgkin's disease monocytes . 295
cells. This observation could be explained by the stimulating effect of PMA on CSF release from monocytes, and is in agreement with studies of SULLIVAN et al. (21). In contrast to these findings, HDMo were unable to enhance CFU-dG growth stimulation after incubation with PMA. This phenomenon was not observed when indomethacin was incorporated into the culture, which seems to indicate that an increased production of prostaglandins by PMA-treated HDMo was a possible cause of this disparity. At higher cell concentrations there was a no difference in the CFU-dG growth promoting effect between resting and PMA-treated HDMo. This may indicate that HDMo, under the experimental conditions, did not respond to PMA stimulation. Of interest was the finding that the increase in cloning efficiency in experiments with HDMo and indomethacin was higher than in comparable investigations with nMo, suggesting that HDMo produce more PGEz. This observation is in agreement with previously reported data (9, 10). The discrepancies between normal and HD monocytes were confirmed in cross experiments, where allogeneic normal and HD bone marrows were used as target cells (Fig. 3). In addition, the results of these investigations provide evidence that accessory cells from HD marrow could not account for this altered HDMo function. The exact mechanism of different CFU-dG growth promoting effect in response to PMA between normal and HDMo remains obscure. It is well established that monocytes in HD produce markedly increased levels of PGE z (9, 10). Furthermore, as mentioned above, these cells reveal an inability to respond to protein kinase C activators. Taken together, these data may suggest that signal transduction and consequently responsiveness of HDMo might be essentially changed. However, the mechanism underlying this altered response is still unclear. It is possible, according to the recent concept of HD ethiopathology, that the growth of malignant cells and local as well as systemic release of some cytokines (interleukin-l, TNF, interferon) modulate activation and proliferation of several nonmalignant cells, mostly lymphocytes and monocytes/macrophages (22). The cooperation of these cells in the immune response and in the regulation of hematopoiesis is well known. Thus the alteration of the monocytes function in HD could be indirectly mediated by some accessory cells and/or their products. In the present study marrow and blood cell populations were not homogeneous. Monocyte-enriched mononuclear cells were contaminated mainly by granulocytes. It is known that these cells produce lactoferrin, a CSA-release inhibitor (23), however, it seems improbable that this factor played any significant role in our studies since, as we showed previously, polymorphonuclears have no direct effect on granulocytic progenitors growth in diffusion chamber culture (24). It is possible that the admixture of granulocytes might diminish CSA production to some degree. However, this effect is negligible since the number of granulocytic colonies at higher
296 .
J.
HANSZ, K. SAWINSKI, and T. WOZNY
concentrations of monocyte-enriched cells in experiments with indomethacin was very high, and could not be enhanced by any further increase in Mo number. The present studies were performed with monocytes only from patients with advanced HD. At these disease stages, the demonstrated monocyte preactivation has no dependence on the clinical/pathological stage, histological type or the presence of constitutional symptoms. Whether the monocytes from patients with more localized disease also display a similar abnormality in the regulation of CFU-dG growth remains to be defined. In conclusion, our results suggest that monocytes from HD patients apart from their defective immune properties, also reveal a functional alteration as granulopoiesis regulating cells, at least in diffusion chamber culture.
References 1. MORAHAN, P. S. 1980. Macrophage nomenclature: Where are we going? J. Reticuloendothe!. Soc. 27: 223. 2. METCALF, D. 1986. The molecular biology and functions of the granulocyte-macrophage colony stimulating factors. Blood 67: 257. 3. KURLAND, J. I., H. E. BROXMEYER, L. M. PELUS, R. S. BOCKMAN, and M. A. S. MOORE. 1978. Role of monocyte-macrophage-derived colony stimulating factor and prostaglandin E in the positive and negative feedback control of myeloid stem cell proliferation. Blood 52: 388. 4. BROXMEYER, H. E., L. JULIANO, L. Lu, E. PLATZER, and B. DUPONT. 1984. HLA-DR human histocompatibility leukocyte antigen restricted lymphocyte-monocyte interactions in the release from monocytes of acidic isoferritins that suppress hematopoietic progenitors cells. J. Clin. Invest. 73: 939. 5. MATTHEWS, N. 1981. Production of an antitumor cytotoxin by human monocytes. Immunology 44: 135. 6. URBANITZ, D., I. FECHNER, and R. GROSS. 1975. Reduced monocyte phagocytosis in patients with advanced Hodgkin's disease and lymphosarcoma. Klin. Wschr. 53: 437. 7. LEB, L., and J. A. MERRITT. 1978. Decreased monocyte function in patients with Hodgkin's disease. Cancer 41: 1794. 8. NAGAI, H., R. I. FISHER,]. COSSMAN, and].]. OPPENHEIM. 1986. Decreased expression of class II major histocompatibility antigens on monocytes from patients with Hodgkin's disease. J. Leucocyte Bio!. 39: 313. 9. GOODWIN, J. S., R. P. MESSNER, A. D. BANKHURST, G. T. PEAKE, J. H. SAIKI, and R. WILLIAMS. 1977. Prostaglandin producing suppressor cells in Hodgkin's disease. N. Eng!. J. Med. 297: 963. 10. PASSWELL, J., M. LEVANON, J. DAVIDSOHN, and B. RAMOT. 1983. Monocyte PGE 2 secretion in Hodgkin's disease and its relation to decreased cellular immunity. Clin. Exp. Immuno!. 51: 61. 11. PARK, B. H., S. M. FIKRIG, and E. M. SMITHWICK. 1968. Infection and nitrobluetetrazolium reduction by neutrophils. Lancet 2: 532. 12. COHEN, H. J., and M. E. CHOVANIEC. 1978. Superoxide generation by digitonin stimulated guinea pig granulocytes. J. Clin. Invest. 61: 1081. 13. GORDON, M. Y., and N. M. BLACKETT. 1975. Stimulation of granulocytic colony formation in agar diffusion chambers implanted in cyclophosphamide pretreated mice. Brit. J. Cancer 32: 51.
CSA release by Hodgkin's disease monocytes . 297 14. JACOBSEN, N., H. E. BROXMEYER, E. GROSSBARD, and M. A. S. MOORE. 1978. Diversity of human granulopoietic precursor cell: separation of cells that form colonies in diffusion chambers (CFU-D) from populations of colony forming cells in vitro (CFU-C) by velocity sedimentation. Blood 52: 221. 15. NISKANSEN, E., T. OLOFSSON, and M. J. CLINE. 1979. Hemopoietic precursor cells in human peripheral blood. Am J. Hematol. 7: 201. 16. SHADDUCK, R. K., G. PIGOLI, A. WAHEED, A. CARSTEN, and E. CRONKITE. 1986. Regulation of diffusion-chamber granulopoiesis by colony stimulating factor. Exp. Hematol. 14: 812. 17. GENTILE, P. S., and L. M. PELUSo 1987. In vivo modulation of myelopoiesis by prostaglandin E 2 • II. Inhibition of granulocyte-monocyte progenitor cell (CFU-GM) cell-cycle rate. Exp. Hematol. 15: 119. 18. Lu, L., D. WALKER, C. D. GRAHAM, A. WAHEED, R. K. SHADDUCK, and H. E. BROXMEYER. 1988. Enhancement of release from MHC class II antigen-positive monocytes of hematopoietic colony stimulating factors CSF-1 and G-CSF by recombinant human tumor necrosis factor-alpha: synergism with recombinant human interferon-gamma. Blood 72: 34. 19. CASTAGNA, M. 1987. Phorbol esters as signal transducers and tumor promoters. BioI. Cell 59: 3. 20. YASHRUTI, A. A., K. BERJESTEH, and S. M. TAFFET. 1987. Isolation of 12-0-tetradecanoylphorbol-13-acetate-resistant mutants of a macrophage-like cell line: evidence for induction by 12-0-tetradecanoylphorbol-13-acetate of a non colony stimulating factor. Cancer Res. 47: 2777. 21. SULLIVAN, R., R. A. BRODIE, N. E. LARSEN, P. J. GANS, and L. A. MCCARROLL. 1984. The effects of tumor-promoting phorbol esters on human granulopoiesis in vitro. Blood 64: 526. 22. AGHAI, E. 1986. Hodgkin's disease: malignancy, inflammation and abnormal immunity. Leuk. Res. 10: 1267. 23. BROXMEYER, H. E., A. SMITHYMAN, R. R. EGER, P. A. MEYERS, and M. DESOUSA. 1978. Identification of lactoferrin as the granulocyte-derived inhibitor of colony-stimulating activity (CSA) production. J. Exp. Med. 48: 1052. 24. HANSZ, J., T. WOZNY, J. ]URKOWIECKA, K. SAWINSKI, and O. W. MENSAH. 1990. Polymorphonuclear neutrophils do not affect the clonal growth of granulocyte progenitors in vivo in diffusion chamber culture (in Polish). Acta Haematol. Pol. 21: 66. Prof. Dr. JANUSZ HANSZ, Department of Hematology, Academy of Medicine, 8/12 Szkolna Str., 61-833 Poznan, Poland
Department of Hematology, Academy of Medicine, Poznan, Poland
Impaired Release of Colony Stimulating Activity by Monocytes from Hodgkin's Disease in Response to Phorbol Myristate Acetate Activation JANUSZ HANSZ, KRZYSZTOF SAWIN-SKI, and TOMASZ WOZNY
Received July 10, 1989· Accepted in Revised Form May 21,1990
Abstract We assessed the humoral effect of resting and phorbol esters preincubated monocytes from Hodgkin's disease patients (HDMo) and healthy subjects (nMo), on granulocyte progenitors (CFU-dG) growth using a double diffusion chamber technique. The release of colony stimulating activity and indomethacin-dependent inhibitors by resting HDMo and nMo was found to be cell-concentration dependent. However, phorbol myristate acetate preincubated HDMo (PMA-HDMo) in contrast to nMo at low concentrations (2.5 x 104 ) were unable to increase the CFU-dG growth stimulation. On the other hand, at a higher cell number (5 x 104 ), phorbol treated HDMo stimulated the myeloid colony formation, whereas nMo suppressed the CFU-dG proliferation. Further enhancement of HDMo and nMo concentrations induced a pronounced inhibition of CFU-dG-derived colony formation, caused by an increased PGE 2 production. After incubation with the cyclooxygenase inhibitor-indomethacin, PMA-HDMo showed considerably more granulocyte colony formation than nMo. Our results suggest that the observed abnormalities in the function of HDMo could be associated with an excessive production of PGE 2 and a general dysfunction of these cells in Hodgkin's disease.
Introduction Monocytes (Mo) playa crucial role in the regulation of hematopoiesis, at least in culture. Directly and in cooperation with other cells, they produce humoral factors that stimulate or suppress the hematopoietic precursor proliferation (1). Monocyte derived colony stimulating factors (CSF), like other CSFs, are glycoproteins essential for myeloid colony formation (2). Monocytes also release potent inhibitors of granulocyte progenitor growth, such as prostaglandin E (3), acidic isoferritins (4), and tumor necrosis factor
(TNF) (5). Abbreviations: CFU-dG = colony forming unit-granulocyte (diffusion chamber); CSA = colony stimulating activity; FCS = fetal calf serum; HBSS = Hanks' balanced salt solution; HD = Hodgkin's disease; Mo = monocytes; NBT = nitro blue tetrazolium; PBS = phosphate-buffered saline; PMA = phorbol12-myristate 13-acetate; TNF = tumor necrosis factor; PGE2 = prostaglandin E2 .
CSA release by Hodgkin's disease monocytes . 289
In Hodgkin's disease (HD) the monocytes reveal many abnormalities, e.g. reduced phagocytic activity (6), decreased chemotactic response (7), diminished expression of class II MHC antigens (8) and an increased prostaglandin production (9, 10). These abnormalities may be of great significance for the impaired immunity in HD. Little is known, however, whether HD monocytes demonstrate a normal or defective function in the regulation of hematopoiesis. In the present study, we were investigating the effects of resting and PMA-activated HDMo on granulocyte progenitor growth and subsequently found that these cells, being already preactivated, revealed an altered response to phorbol stimulation and an abnormal modulation of myeloid colony formation in diffusion chamber culture. Materials and Methods Patients Peripheral blood (PB) and bone marrow (BM) samples were collected from 15 patients with HD in the clinical/pathological stage III or IV. The specimens for control experiments were obtained from 15 hematologically normal individuals undergoing cardiac surgery. Both PB and BM samples were gathered after informed consent had been obtained from the patients in accordance with the guidlines of the Academy of Medicine Committee, for protection of human subjects. Cell separation Bone marrow mononuclear cells isolated by Ficoll/Hypaque (specific gravity 1.077; Pharmacia, Uppsala, Sweden) density gradient centrifuging at 400 g for 15 min, after washing three times, were then resuspended in McCoy's medium (Flow, U.K.) containing 20 % fetal calf serum (FCS, Calbiochem, Switzerland), 200 U/ml penicillin G and 200 U/ml streptomycin. The viability of cells, assessed by Trypan Blue exclusion, exceeded 96 %. Monocytes were separated from PB. The blood was diluted in an equal volume of Hanks' Balanced Salt Solution (HBSS, Biomed, Poland) and the mononuclear cells were isolated by Ficoll/Hypaque density gradient centrifuging at 400 g for 40 min. Cells from the interphase, after washing three times and resuspending in McCoy'S medium supplemented with 20 % FCS in a final concentration of 106 cells/ml, were incubated in plastic Petri dishes at 37°C in 5 % CO 2 • After 1.5 h, the non-adherent cells were removed by gently washing the dishes with HBSS. The adherent cells were recovered by scraping and then suspended in McCoy's medium enriched with FCS and antibiotics in the same concentrations as above. The adherent cell suspension consisted of monocytes (mean 76 %), neutrophils (20 %), eosinophils (2 %), and lymphocytes (2 %) as determined by the May-Grunwald-Giemsa method and by the nonspecific esterase staining. In some experiments, monocytes were incubated for 5 min, at 37°C with phorbol 12myristate 13-acetate (Sigma, USA) in a final concentration of 1 flg/ml of McCoy's medium containing 106 cells/m!. After incubation, monocytes were washed twice and resuspended in McCoy's medium supplemented with 20 % FCS and antibiotics. In the control experiments, the cells were incubated under the same conditions, except without phorbol myristate acetate (PMA). The viability of monocytes each time exceeded 90 %. Nitroblue tetrazolium (NBT) reduction test The NBT reduction test was performed following methods used by PARK et al. (11). Monocyte suspension (5 x 105 /ml PBS) was mixed with an equal volume of 0.2 % nitro blue
290 . ]. HANSZ, K. SAWINSKI, and T. WOZNY Table 1. NBTa reduction test of monocytes from normal subjects and patients with Hodgkin's disease Material
Monocytes resting
PMA-treated b
Healthy subjects
22 ± Se
61 ± 7
Hodgkin's disease patients
4S ± 10
57 ± 11
a NBT - nitro blue tetrazolium monocytes incubated with phorbol12-myristate 13-acetate (2 !-Ig/m1/106 cells, 5 min., 37°C) C percent of NBT-positive cells (mean ± standard deviation of six separate experiments)
b
tetrazolium (NBT, Sigma, USA) in 0.15 M NaCI. The cells were incubated for 30 min at 37°C and then again for 30 min at IS dc. The percentage of formazan containing cells was determined on glass slides stained by the Pappenheim method.
Superoxide anion (Oi) production The O 2 production was estimated by the method described by COHEN and CHOVANIEC (12). Briefly, 0.1 ml of monocyte suspension (5 x 105 cells/ml PBS) was mixed with 0.3 ml of cytochrom C solution (2 mg/ml; WSiSz, Krakow, Poland). After incubation (15 min, 3rq superoxide dismutase (2 mg/ml, Sigma, USA) was added. The extinction of supernatant was measured at 550 nm wave length. The control sample was prepared in the same manner but without monocytes. 0;; production was calculated in nmol of reduced cytochrom C for 106 monocytes.
Diffusion chamber culture The agar diffusion chamber technique used for granulocyte colony formation was a modification of the method of GORDON and BLACKETT (13). Briefly, the diffusion chambers (DC) used in this study, consisted of two 400 !-II compartments separated by a membrane filter (Sartorius, pore size 0.45 !-1m). The chambers were covered on one side by a membrane filter and sealed on the other by a glass slide. Bone marrow cells (2 x 105/0.4 ml), resuspended in McCoy's medium with FCS and antibiotics in 0.3 % agar (Difco, USA), were introduced into the DC compartment covered on both sides by a filter. Monocytes placed in the second DC-compartment were suspended in the same medium, but with 0.5 % agar. In some of the experiments, together with monocytes, indomethacin (Merck, FRG) was incorporated into the chambers at a final concentration of Table 2. Superoxide anion production by resting and phorbol myristate acetate preincubated normal and Hodgkin's disease monocytes Material
Monocytes resting
Healthy subjects Hodgkin's disease patients
6.6 ± 2.Sb 15.2 ± 5.7
PMA-treateda 19.9±5.5 17.l±7.2
a monocytes incubated with phorbol12-myristate 13-acetate (2 !-Ig/m1/106 cells, 5 min., 37°C) 6 b nmol superoxide anions!10 cells (mean ± standard deviation of six separate experiments)
CSA release by Hodgkin's disease monocytes . 291 1.7 mmolli. After gelling of agar at room temperature, the DCs were implanted into the peritoneal cavity of rats (female, Wi star strain, weighing 200-300 g) which had been irradiated with X-ray (6.0 Gy) 2 h prior to implantation. The cultures were performed in quadruplicate for each set of experiments. On day 9 the number of colonies (> 20 cells) was determined. The cellular composition of colonies was determined on slides stained by the May-Griinwald-Giemsa method and also evaluated for myeloperoxidase, naphthyl-ASD chloroacetate esterase and acid a-naphthylacetate esterase. The colonies consisted of myeloblasts (20 %), myelocytes (39 %), metamyelocytes (25 %) and mature granulocytes (16 %). No significant difference between the cellular composition of colonies grown from normal and HD marrow was seen. Results were analysed using Student's t test. Differences were considered statistically significant if p < 0.05.
Results
NBT reduction test and superoxide anion production in resting and PMApreincubated normal (nMo) and Hodgkin's disease monocytes (HDMo)
In comparison with resting nMo, after incubation with PMA, an increased number of cells contained formazan and revealed an augmented superoxide anion production (p < 0.05; Table 1). In contrast to nMo, resting HDMo already displayed an enhanced superoxide production and an increased formazan content (p < 0.05). Preincubation with PMA did not change these characteristics (p > 0.05; Table 2).
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MONOCYTES Figure 1. Effect of normal monocytes (Mo) on autologous marrow granulocytic precursors proliferation in double diffusion chamber culture. Resting Mo (e--e), Mo incubated with 1 f.lg/ml phorbol12-myristate 13-acetate (PMA-Mo) (0---0), PMA-Mo and 1.7 mmolll indomethacin (0---0). Each point represents a mean ± standard deviation from four experiments done in quadruplicate. The results are expressed as a percentage of control (colony formation without monocytes).
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Figure 2. Effect of monocytes from Hodgkin's disease patients (HDMo) on autologous marrow granulocytic precursors proliferation in diffusion chamber culture. Resting HDMo (e--e), HDMo incubated with 1 flg/ml phorbol12-myristate 13-acetate (PMA-HDMo) (0---0), PMA-HDMo and, 1.7 mmol!l indomethacin (0---0). Each point represents a mean ± standard deviation from four experiments done in quadruplicate. The results are expressed as a percentage of control (colony formation without monocytes).
Effect of normal monocytes (nMo) on autologous marrow granulocytic precursors prollferation In control cultures (without monocytes), the marrow granulocyte progenitors formed 27 ± 3 colonies (mean ± SD from four experiments done in quadruplicate). In experiments with monocytes, the myeloid colony formation was dependent on the number of monocytes introduced into the chambers (Fig. 1). The highest cloning efficiency was observed at 5 x 104 nMo and the colony number (50 ± 7) increased almost 85 % over the control value (p < 0.01). At higher Mo concentrations the colony count was diminished. In cultures with 1.0 x 105 nMo the myeloid colony formation was within the range of the control value (p > 0.05). The most significant stimulating effect of PMA-preincubated nMo was observed at a lower concentration of these cells (2.5 x 104 /chamber) in comparison with resting nMo. With higher Mo numbers the colony count was markedly lower (p < 0.05). The inhibition of CFU-dG growth at 5 x 10\ 7.5 X 10 4 and 1.0 x 105 nMo was entirely abolished when indomethacin, together with Mo suspension, was introduced into the chambers and the myeloid colony formation was within the range observed for maximum stimulation in experiments performed with resting monocytes (p> 0.05). Resting and activated nMo exerted a similar CFU-dG growth enhancing effect independently, whether normal or HD marrow cells were used (Fig. 3A).
CSA release by Hodgkin's disease monocytes . 293
Effect of Hodgkin's disease monocytes (HDMo) on autologous marrow granulocytic precursors proliferation In control experiments (without monocytes), the cloning efficiency of HD marrow cells was significantly lower compared with the normal controls (19 ± 3; P < 0.05). Resting HDMo promoted autologous CFU-dG growth like the monocytes from healthy subjects (Fig. 2). Also the greatest stimulating effect was detected at 5 x 104 Mo and the colony number (43 ± 6) exceeded the control value by greater than 90 % (p < 0.01). In contrast to the results obtained with PMA-treated nMo, however, PMA-preincubated HDMo, at a lower concentration (2.5 x 104 ), stimulated CFU-dG-derived colony formation to a much lesser degree (p < 0.05). The maximum increase in CFU-dG growth was noted at 5.0 x 104 Mo, similarly as in cultures with unstimulated cells (p> 0.05). Higher monocyte concentration, induced a suppression of the myeloid colony formation in comparison with the effect of resting cells. As in the case of nMo, in the presence of indomethacin, no inhibition of CFU-dG proliferation at higher HDMo concentrations was observed. It should be stressed, that under these experimental conditions, although the increase in cloning efficiency observed in investigations with HDMo was relatively higher than that noted in comparable cultures with nMo, the absolute number of colonies formed was similar (29 ± 5 and 44 ± 11 respectively; p > 0.05). There was no relationship between HDMo functional impairment and the clinical characteristics. However, it should be emphasized that only patients in advanced stages of the disease were studied.
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Figure 3. Effect of resting and phorbol myristate acetate (PMA) preincubated normal (A) and Hodgkin's disease (B) monocytes on allogenous marrow granulocytic precursors proliferation in diffusion chamber culture. (A): normal resting monocytes (e--e) or normal PMAtreated monocytes (0---0) + Hodgkin's disease marrow mononuclear cells. (B): Hodgkin's disease resting monocytes (e--e) or PMA-treated Hodgkin's disease monocytes (0---0) + normal marrow mononuclear cells.
294 .
J. HANSZ, K.
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The effect of resting and PMA-treated HDMo on the growth of marrow CFU-dG from healthy subjects was comparable to that observed in experiments with autologous marrow cells (Fig. 3B).
Discussion In this paper, we are presenting evidence that resting and phorbol treated monocytes from Hodgkin's disease patients and healthy subjects produce growth-promoting and inhibiting activities for granulocyte progenitors, which proliferate and differentiate in diffusion chamber culture. It should be emphasized, that this population of granulocyte progenitors differs essentially from that which gives rise to myeloid colonies in vitro (14, 15). The monocyte effect on autologous marrow granulocytic progenitor growth, both in Hodgkin's disease and in the control, was concentration dependent. Dose response curves indicated that lower Mo numbers induced an increase in cloning efficiency, whereas at higher concentrations a marked suppression of CFU-dG-derived colony formation was observed. These findings are not surprising since monocytes are a potent source of CSF, which is essential for granulocytic colony growth in vitro and in vivo in diffusion chamber culture (2, 16). The decline of the colony formation at higher Mo concentrations was clearly due to prostaglandin release from monocytes, since it was completely abolished under the influence of cyclooxygenase inhibitor-indomethacin (17). This creates an argument against the possible effect of other inhibitors, such as TNF or interferon, which could be released by monocytes and in a peritoneal environment (5, 18). No difference in granulocyte progenitors growth promoting effect of normal or HD monocytes was observed unless PMA-treated cells were tested. Phorbol esters, which are protein kinase C activators, became a useful tool in the study of signal transduction and cell activation (19, 20). The present work has confirmed the stimulating property of PMA on monocytes by demonstrating the changes in super oxide production and in NBT reduction test. Unfortunately, the monocyte suspension used in these experiments was to some degree contaminated by granulocytes and lymphocytes. It seems rather doubtful that these cells could mediate in the PMA activation of monocytes. However, unless a pure population of cells is used, such a possibility can not be ruled out. Of interest was the finding that intact HDMo, in contrast to normal cells, being already activated, were insensitive to further stimulation by PMA. This may indicate that HDMo are unable to respond to protein kinase C activators. The present study clearly shows that monocyte from healthy subjects, after incubation with PMA, revealed their maximum CFU-dG growth promoting effect at lower cell concentrations when compared with resting
CSA release by Hodgkin's disease monocytes . 295
cells. This observation could be explained by the stimulating effect of PMA on CSF release from monocytes, and is in agreement with studies of SULLIVAN et al. (21). In contrast to these findings, HDMo were unable to enhance CFU-dG growth stimulation after incubation with PMA. This phenomenon was not observed when indomethacin was incorporated into the culture, which seems to indicate that an increased production of prostaglandins by PMA-treated HDMo was a possible cause of this disparity. At higher cell concentrations there was a no difference in the CFU-dG growth promoting effect between resting and PMA-treated HDMo. This may indicate that HDMo, under the experimental conditions, did not respond to PMA stimulation. Of interest was the finding that the increase in cloning efficiency in experiments with HDMo and indomethacin was higher than in comparable investigations with nMo, suggesting that HDMo produce more PGEz. This observation is in agreement with previously reported data (9, 10). The discrepancies between normal and HD monocytes were confirmed in cross experiments, where allogeneic normal and HD bone marrows were used as target cells (Fig. 3). In addition, the results of these investigations provide evidence that accessory cells from HD marrow could not account for this altered HDMo function. The exact mechanism of different CFU-dG growth promoting effect in response to PMA between normal and HDMo remains obscure. It is well established that monocytes in HD produce markedly increased levels of PGE z (9, 10). Furthermore, as mentioned above, these cells reveal an inability to respond to protein kinase C activators. Taken together, these data may suggest that signal transduction and consequently responsiveness of HDMo might be essentially changed. However, the mechanism underlying this altered response is still unclear. It is possible, according to the recent concept of HD ethiopathology, that the growth of malignant cells and local as well as systemic release of some cytokines (interleukin-l, TNF, interferon) modulate activation and proliferation of several nonmalignant cells, mostly lymphocytes and monocytes/macrophages (22). The cooperation of these cells in the immune response and in the regulation of hematopoiesis is well known. Thus the alteration of the monocytes function in HD could be indirectly mediated by some accessory cells and/or their products. In the present study marrow and blood cell populations were not homogeneous. Monocyte-enriched mononuclear cells were contaminated mainly by granulocytes. It is known that these cells produce lactoferrin, a CSA-release inhibitor (23), however, it seems improbable that this factor played any significant role in our studies since, as we showed previously, polymorphonuclears have no direct effect on granulocytic progenitors growth in diffusion chamber culture (24). It is possible that the admixture of granulocytes might diminish CSA production to some degree. However, this effect is negligible since the number of granulocytic colonies at higher
296 .
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HANSZ, K. SAWINSKI, and T. WOZNY
concentrations of monocyte-enriched cells in experiments with indomethacin was very high, and could not be enhanced by any further increase in Mo number. The present studies were performed with monocytes only from patients with advanced HD. At these disease stages, the demonstrated monocyte preactivation has no dependence on the clinical/pathological stage, histological type or the presence of constitutional symptoms. Whether the monocytes from patients with more localized disease also display a similar abnormality in the regulation of CFU-dG growth remains to be defined. In conclusion, our results suggest that monocytes from HD patients apart from their defective immune properties, also reveal a functional alteration as granulopoiesis regulating cells, at least in diffusion chamber culture.
References 1. MORAHAN, P. S. 1980. Macrophage nomenclature: Where are we going? J. Reticuloendothe!. Soc. 27: 223. 2. METCALF, D. 1986. The molecular biology and functions of the granulocyte-macrophage colony stimulating factors. Blood 67: 257. 3. KURLAND, J. I., H. E. BROXMEYER, L. M. PELUS, R. S. BOCKMAN, and M. A. S. MOORE. 1978. Role of monocyte-macrophage-derived colony stimulating factor and prostaglandin E in the positive and negative feedback control of myeloid stem cell proliferation. Blood 52: 388. 4. BROXMEYER, H. E., L. JULIANO, L. Lu, E. PLATZER, and B. DUPONT. 1984. HLA-DR human histocompatibility leukocyte antigen restricted lymphocyte-monocyte interactions in the release from monocytes of acidic isoferritins that suppress hematopoietic progenitors cells. J. Clin. Invest. 73: 939. 5. MATTHEWS, N. 1981. Production of an antitumor cytotoxin by human monocytes. Immunology 44: 135. 6. URBANITZ, D., I. FECHNER, and R. GROSS. 1975. Reduced monocyte phagocytosis in patients with advanced Hodgkin's disease and lymphosarcoma. Klin. Wschr. 53: 437. 7. LEB, L., and J. A. MERRITT. 1978. Decreased monocyte function in patients with Hodgkin's disease. Cancer 41: 1794. 8. NAGAI, H., R. I. FISHER,]. COSSMAN, and].]. OPPENHEIM. 1986. Decreased expression of class II major histocompatibility antigens on monocytes from patients with Hodgkin's disease. J. Leucocyte Bio!. 39: 313. 9. GOODWIN, J. S., R. P. MESSNER, A. D. BANKHURST, G. T. PEAKE, J. H. SAIKI, and R. WILLIAMS. 1977. Prostaglandin producing suppressor cells in Hodgkin's disease. N. Eng!. J. Med. 297: 963. 10. PASSWELL, J., M. LEVANON, J. DAVIDSOHN, and B. RAMOT. 1983. Monocyte PGE 2 secretion in Hodgkin's disease and its relation to decreased cellular immunity. Clin. Exp. Immuno!. 51: 61. 11. PARK, B. H., S. M. FIKRIG, and E. M. SMITHWICK. 1968. Infection and nitrobluetetrazolium reduction by neutrophils. Lancet 2: 532. 12. COHEN, H. J., and M. E. CHOVANIEC. 1978. Superoxide generation by digitonin stimulated guinea pig granulocytes. J. Clin. Invest. 61: 1081. 13. GORDON, M. Y., and N. M. BLACKETT. 1975. Stimulation of granulocytic colony formation in agar diffusion chambers implanted in cyclophosphamide pretreated mice. Brit. J. Cancer 32: 51.
CSA release by Hodgkin's disease monocytes . 297 14. JACOBSEN, N., H. E. BROXMEYER, E. GROSSBARD, and M. A. S. MOORE. 1978. Diversity of human granulopoietic precursor cell: separation of cells that form colonies in diffusion chambers (CFU-D) from populations of colony forming cells in vitro (CFU-C) by velocity sedimentation. Blood 52: 221. 15. NISKANSEN, E., T. OLOFSSON, and M. J. CLINE. 1979. Hemopoietic precursor cells in human peripheral blood. Am J. Hematol. 7: 201. 16. SHADDUCK, R. K., G. PIGOLI, A. WAHEED, A. CARSTEN, and E. CRONKITE. 1986. Regulation of diffusion-chamber granulopoiesis by colony stimulating factor. Exp. Hematol. 14: 812. 17. GENTILE, P. S., and L. M. PELUSo 1987. In vivo modulation of myelopoiesis by prostaglandin E 2 • II. Inhibition of granulocyte-monocyte progenitor cell (CFU-GM) cell-cycle rate. Exp. Hematol. 15: 119. 18. Lu, L., D. WALKER, C. D. GRAHAM, A. WAHEED, R. K. SHADDUCK, and H. E. BROXMEYER. 1988. Enhancement of release from MHC class II antigen-positive monocytes of hematopoietic colony stimulating factors CSF-1 and G-CSF by recombinant human tumor necrosis factor-alpha: synergism with recombinant human interferon-gamma. Blood 72: 34. 19. CASTAGNA, M. 1987. Phorbol esters as signal transducers and tumor promoters. BioI. Cell 59: 3. 20. YASHRUTI, A. A., K. BERJESTEH, and S. M. TAFFET. 1987. Isolation of 12-0-tetradecanoylphorbol-13-acetate-resistant mutants of a macrophage-like cell line: evidence for induction by 12-0-tetradecanoylphorbol-13-acetate of a non colony stimulating factor. Cancer Res. 47: 2777. 21. SULLIVAN, R., R. A. BRODIE, N. E. LARSEN, P. J. GANS, and L. A. MCCARROLL. 1984. The effects of tumor-promoting phorbol esters on human granulopoiesis in vitro. Blood 64: 526. 22. AGHAI, E. 1986. Hodgkin's disease: malignancy, inflammation and abnormal immunity. Leuk. Res. 10: 1267. 23. BROXMEYER, H. E., A. SMITHYMAN, R. R. EGER, P. A. MEYERS, and M. DESOUSA. 1978. Identification of lactoferrin as the granulocyte-derived inhibitor of colony-stimulating activity (CSA) production. J. Exp. Med. 48: 1052. 24. HANSZ, J., T. WOZNY, J. ]URKOWIECKA, K. SAWINSKI, and O. W. MENSAH. 1990. Polymorphonuclear neutrophils do not affect the clonal growth of granulocyte progenitors in vivo in diffusion chamber culture (in Polish). Acta Haematol. Pol. 21: 66. Prof. Dr. JANUSZ HANSZ, Department of Hematology, Academy of Medicine, 8/12 Szkolna Str., 61-833 Poznan, Poland
