Action of inhibitor released from neutrophils and leukemic blast cells PETER R. GALBRAITH,* MD, FRCP[CI; Lois J. CooKE;t FRASER L. BAKER4 PH D
The concept that polymorphonuclear leukocytes, or neutrophils, play a role in feedback control of granulopolesis has been supported by the finding in bone marrow culture studies that mature neutrophils inhibited formation of granulocytic colonies. The study described in this paper was done to investigate the mechanisms involved. With the use of a modified assay it was found that mature neutrophils released factors that reduced the proliferation of colony-forming cells in cultures stimulated by cell-free colony-stimulating factor. In myeloproliferative and myelodysplastic disorders the amount of inhibitor released by the neutrophils varied greatly. Leukemic blast cells also released inhibitor, and in some cases the amount released per cell was greater than tha amount released from normal mature neutrophils. The inhibitory factors released from the neutrophils differed from those previously described in the literature in terms of mode of action and apparent molecular size. La notion voulant que les leucocytes polymorphonucleaires, ou neutrophiles, jouent un r6le dans le contr6le par retroaction de Ia granulopolese s'est vu soutenue par l'observation dans les 6tudes sur Ia culture des cellules de moelles osseuses que les neutrophiles a maturite inhibaient Ia formation des cellules granulocytaires. L'etude qui fait l'objet de cette publication a et6 falte dans le but d'investiguer les m6canismes impliques. A l'aide d'une technique modifiee on a trouve que les neutrophiles a maturite liberent des facteurs aul r6duisent Ia proliferation des cellules formant des colonies dans des cultures stimulees par un facteur libre de cellules qui favorise Ia formation de colonies. Dans les maladies my6loproliferatives et les myelodysplasies les quantites d'inhibiteur liber6es par les neutrophiles ont vari6 consid6rablement. Les cellules leucemiques blastiques liberent aussi de cet From the department of medicine, Queen's University, Kingston *Profe.or of medicine tSenior technician, division of hematology .Research associate Reprint requests to: Dr. Peter R. Gaibraith, 102 Stuart St., Ste. 113, Kingston, Ont. K7L 3N6
inhibiteur, et dans certains cas Ia quantite lib6r6e par cellule surpassait Ia quantit6 lib6r6e par des neutrophiles normaux a maturit6. Les facteurs inhibiteurs lib6res par les neutrophiles differalent de ceux d6jA d6crits dans Ia litterature par leur mode d'action et leur poids moleculaire apparent.
End-product inhibition is an important regulatory mechanism in many biologic systems. The concept that polymorphonuclear leukocytes, or neutrophils, exert feedback control over granulopoiesis has been suggested by the results of in vitro studies that indicate that neutrophils inhibit the formation of granulocytic colonies.'4 Such studies may have clinical relevance since some leukemic neutrophils appear not to inhibit,34 and remission of acute leukemia has occurred following treatment with granulocytic chalone.6 In this paper we describe our study that showed that normal neutrophils released factors that inhibited the proliferation of granulocyte-committed colony-forming cells in cultures stimulated by cell-free colonystimulating factor. These factors differed from those previously described in molecular size and site of action. Furthermore, the amount of inhibitor released by leukemic neutrophils and blast cells varied greatly. Methods Collection of cells Venous blood samples and bone marrow aspirates were collected from informed healthy volunteers and patients with various hematologic disorders, as described previously,2 in tubes containing 0.5 mL of citratephosphate-dextrose solution USP (Fenwal Laboratories, division of Baxter Travenol Laboratories of Canada Ltd., Malton, Ont.) as an anticoagulant and 3% dextran to accelerate the sedimentation of erythrocytes. The nucleated-cell-rich suspensions were washed three times in
CMRL 1066 tissue culture medium (Grand Island [New York] Biological Company) and then subjected to cell separations based on cell density in Ficoll® (density 1.070 g/mL; dissolved in CMRL 1066 tissue culture medium) (Pharmacia Fine Chemicals, Uppsala, Sweden) and adhesiveness to plastic.3'7 The following fractions were collected: 1. The light-density mononuclear leukocyte fraction of normal blood 3,8 leukucyte suspensions. This pro vided a mononuclear leukocyte suspension containing monocytes, which was used in the preparation of colony-stimulating factor. 2. The high-density fraction of blood leukocytes.' This yielded cell suspensions highly enriched with neutrophils. 3. The nonadherent light-density nucleated cell fraction of normal human bone marrow cells,8 to serve as the source of target colony-forming cells. These were used in all the experiments described. Preparation of colony-stimulating factor Mononuclear-leukocyte-conditioned medium or serum provided cellfree colony-stimulating factor for all experiments. They were prepared respectively by incubating 2 x 10. normal blood mononuclear leukocytes per millilitre at 370C for 5 to 7 days in tissue culture medium containing 30% normal human serum heated at 580C for 150 minutes8 or in 100% neutropenic serum obtained from patients with acute myelomonocytic leukemia and severe neutropenia (neutrophil count less than 0.6 x 109/L). Comparable results were obtained with the two preparations. Neutropenic serum was used initially to minimize contamination by inhibitor released from neutrophils; inhibitory activity is destroyed by heat treatment of normal human serum.8
CMA JOURNAL/MARCH 3, 1979/VOL. 120 545
The two preparations were assayed batch of colony-stimulating factor. to determine the least concentration The assay cultures were virtually dethat promoted maximum colony for- pleted of monocytes7 so as to prevent mation. This concentration was then endogenous elaboration of colonyused in all the experiments described. stimulating factor. The reduced content of neutrophils"3 made the assay Preparation ci inhibitor cultures more sensitive to both Inhibitor from normal blood or colony-stimulating factor and inhibibone marrow cell suspensions was tors.9'10 prepared by incubating 2 x 106 neuIn all experiments aliquots of trophils per millilitre in CMRL 1066 CMRL 1066 tissue culture medium tissue culture medium for 10 minutes, conditioned by specified leukocyte and then removing the cells by Milli- populations were assayed at a 10% pore filtration (filter pore size 0.45 dilution tor their inhibition of colony p.m). The filtrate, termed neutrophil- formation in cultures stimulated by conditioned medium, was frozen and cell-free colony-stimulating factor. In stored at -25 0C. Initially the neutro- patient studies a dilution curve for phils were incubated at 370C, but neutrophil-conditioned medium presubsequently it was found that incu- pared from normal blood was used bation at room temperature (22 0C) to establish the validity of the assay yielded similar results and was much - that is, that the inhibitory effect easier. However, in individual expe- of this medium was concentrationriments the temperature at which the dependent - and the potency of a conditioned medium was prepared 10% dilution of this medium was was kept constant and is specified. assessed simultaneously with that of Inhibitor was prepared from leu- a 10% dilution of neutrophil-condikemic blood or bone marrow cell tioned medium prepared from leususpensions as described above. kemic blood. When the suspensions contained less Since the leukemic-blast-cell-conthan 90% blast cells or more than 5% neutrophils, density separation in Ficoll® was used to enrich for 40blast cells and remove neutrophils. Cell cultures The single-layer semisolid agar culture system was used throughout the study.1'3 Each 1-mL culture contained 0.3% agar to minimize dispersal of colonies. The culture medium, described previously,2'3 was modified to contain 30% normal human serum heated at 580C for 150 minutes instead of fetal calf serum, so as to provide appropriate supplementation with "essential serum growth factors" (or permissive factors), with retention of sensitivity to the action of both the stimulatory and the inhibitory molecules under investigation.8 Experimental design All agar cultures contained 0.75 to 1 x 1 0.' nonadherent light-density marrow cells. To provide a constant source of colony-forming cells in each experiment, we used marrow from one healthy individual and a single
ditioned media were not all studied simultaneously, the results were normalized to show the relative inhibition of colony formation by the two types of conditioned medium. Colony counts Colonies were counted on triplicate plates after 7 days of incubation. In control cultures aggregates of 20 or more cells were considered as colonies, partly on the basis of precedent11'13 and partly on the basis of the results (unpublished) of our preliminary studies, which showed that, in cultures stimulated by increasing concentrations of colony-stimulating factor, when a single plate was removed daily from the incubator and scored for aggregate number and aggregate size, an increase was found in aggregate number with time that was dependent on the concentration of colony-stimulating factor. Aggregates of only 3 to 5 cells appeared on day 2, reached a maximum number on day 3 or 4, and decreased exponentially thereafter. Colonies of 20 or more cells appeared on days 6, 7
No incubation '. with neutrophils 30
Colonies per 20 plate (mean ± SEM)
4
I0-
0 5
10
30
60
Time (minutes)
120
FIG. 1-Release of inhibitor by 2 X 10 neutrophils per millilitre incubated in CMRL 1066 tissue culture medium as function of time of incubation. At end of incubation cell suspensions underwent Millipore filtration (filter pore size 0.45 .m) and 0.1 mL of cefl-free filtrate was added to cultures of nonadherent light-density nucleated cell fractions of normal bone marrow cells. AU cultures were stimulated by 5% mononuclear-leukocyte-conditioned serum, which provided cell-free colony-stimulating factor.
546 CMA JOURNAL/MARCH 3, 1979/VOL. 120
and 8, and thereafter decreased in dilution. The higher than predicted Stability of inhibitor in neutrophilnumber but increased in size. More activity of inhibitor in the dialysis conditioned medium than 95% of colonies present on day fluid suggests that the dialysis memStability was assessed by assaying 7 were peroxidase-positive granulo- brane separated inhibitor from larger the activity of neutrophil-conditioned cytic clones. In cultures with added molecules that would have influenced medium after incubation at 370C inhibitor, colony size was reduced the stability or action of the lower- (Fig. 4). Little activity was detected by approximately 50%. Therefore, molecular-weight inhibitor (Fig. 3). after 72 hours. Inhibitory activity was aggregates of fewer than 20 cells were considered as colonies. This, in effect, biases our data against the hypotheses being tested. I No inhibitor 200Results Kinetics of inhibitor released ISOfrom neutrophils The rate of inhibitor release from Colonies ioo. normal neutrophils was investigated per by assaying aliquots of 10% neutroplate 140 phil-conditioned medium harvested at intervals after initiation of cul- (mean ± SEM) ture. Assuming minimal cell damage 120. during Millipore filtration, the results shown in Fig. 1 indicate that inhibI00. itor was rapidly released, and that inhibitory activity in the medium was maximal within 5 minutes.
Sensitivity of assays Individual experiments varied with respect to the sensitivity of assay cultures to the addition of normal neutrophil-conditioned medium. In all assays inhibition was linearly related to the dilution of the medium. In the most sensitive assays a biphasic inhibitor dilution curve was obtained (Fig. 2): at dilutions of 1:4096 to 1:1024 marked inhibition occurred, and at lower dilutions the slope of the curve decreased. Clearly the assay is relatively insensitive to small differences in inhibitor concentration, but larger differences can be appreciated.
I
4096
024
I
256
64
16
4
Dilution of neutrophil.-conditioned medium FIG. 2-Concentration-dependent effects of inhibitor in neutrophil-conditioned medium diluted with CMRL 1066 tissue culture medium and added (0.1 mL per 1-mL culture) to cultures stimulated by 2% mononuclear-leukocyte-conditioned medium.
Control 120 Neutrophi 1-conditioned medium . 100 .4
Undialysed 10% 0.1%
Dialysed 10%
Dialysis fluid 10%
(0
2 80 0) (0 ..
Effect of dialysis on inhibitory activity We dialysed 10 mL of neutrophilconditioned medium harvested at 10 minutes against 1 L of tissue culture medium for 24 hours at 40C. The inhibitor moved freely through the dialysis membrane (Fisher Scientific Co. Ltd., Ottawa, cat, no. 5-667E; pores admit molecules of molecular weight less than 12 000 daltons). The action of inhibitor in the dialysate was that predicted on the basis of
I
16346
60
0) 0. Si) ..
(-)0
40
rI-i
ri-i
FIG. 3-Effect of dialysis on inhibitory activity. After 10 X 10 neutrophils per miflilitre were incubated in CMRL 1066 tissue culture medium for 10 minutes the cell-free conditioned medium was dialysed for 24 hours at 4C (10 mL of conditioned medium against 1000 mL of tissue culture medium) and assayed at a 10% dilution for inhibition of cultures stimulated by 5% mononuclear-leukocyte-conditioned serum. CMA JOURNAL/MARCH 3, 1979/VOL 120 549
retained in neutrophil-conditioned medium stored at -250C for as long as 90 days. Release of inhibitor from abnormal neutrophils The patients selected for study had unequivocal diagnoses established by standard criteria.13 The density separation in Ficoll® ensured that adequate numbers of neutrophils were obtained for study in all patients. Normal neutrophil-conditioned medium was diluted with CMRL 1066 tissue culture medium so as to obtain a control concentration curve to establish the validity of the assay (Fig. 5A). The control and patients' conditioned media were simultaneously assayed at a single concentration (10%). The activity of inhibitors released from control neutrophils was similar, while that of inhibitors released from neutrophils of patients with a variety of hematopoietic stemcell disorders was variable, even within single diagnostic categories (Fig. 5B). Thirteen different leukemic-blastcell-conditioned media were assayed for inhibitory activity at a single concentration (10%). Because normal neutrophil-conditioned medium in various assays reduced colony formation by 20% to 80%, normalized results are presented in Fig. 6. These show that 11 of the 13 media inhibited colony formation. Inhibitor was released to the greatest extent from blast cells obtained from patients whose bone marrow cultures did not form colonies (aggregates of 20 or more cells). In these cases the blast cell medium was as active or more active than normal neutrophilconditioned medium assayed simultaneously. In one such case the blast cell medium was active at a dilution higher than 1:4096, whereas activity was lost in normal neutrophil-conditioned medium diluted to 1:64 (Fig. 7). Discussion Neutrophils release inhibitors with a variety of reported activities.1"'5''1420 The present study is the first to show that low-molecular-weight factors released from normal neutrophils in-
j
40
LU
No inhibitor
LI')
4'
.0 120 0
E
a, BOO
4-) 0 0.
L 80
0) 0. U'
0 0 0 C.)
60 40
-
I Days
2
4
incubation of inhibitor
FIG. 4-Stability of inhibitory activity of neutrophil-conditioned medium. Aliquota of medium were incubated at 37C for various times, then frozen at -20C, and subsequently diluted 1:4 and added in 0.1-mL portions to cultures of nonadherent light-density nucleated cell fractions of bone marrow cells stimulated by 5% mononuclear-leukocyte-conditioned serum.
A.60
Colonies per plate (mean * SEM)
0
/64
.32
"'Is
'8
/4
1/2
Dilution of neutrophil-conditioned medium Neutrophi 1-conditioned medium
B
60-
None
Normal
PV
CML
AML
AMML
Miscellaneous
50Multiple myeloma
Inhibition 40of colony 30formation (mean * SEM)
6 p-thalassemia minor Q Hodgkins disease
. PR
20-
I
I0-
.PR 6Rem 6u U Id
A 1PR
Aplastic anemia
PNH
. Refractory sideroblastic anemia . Refractory anemia Preleukemia
-t
FIG. 5-Effect on colony formation of 0.1 mL of neutrophil-conditioned medium prepared from 2 X 10. neutrophils per millilitre incubated at 22C. In a representative experiment normal conditioned medium was serially diluted to show the effect of different concentrations of inhibitor (panel A). Panel B shows results when media of controls and patients were assayed simultaneously. Results are expressed as the mean reduction (± SEM) in colony number. All cultures were stimulated by 2% mononuclear-leukocyte-conditioned medium. PV = polycythemia vera; CML = chromc myelogenous leukemia; AML = acute myelogenous leukemia; AMML = acute myelomonocytic leukemia; PNH paroxysmal nocturnal hemoglobinuria; PR = partial remission; Rem. = remission; u = untreated.
550 CMA JOURNAL/MARCH 3, 1979/VOL. 120
Colony-formers Neut.
Blast
Cluster-formers Neut.
Blast
No growth Neut.
Blast
'0 0
.0 0 0
0.
______________
been partially resolved, there is variability in the sensitivity of the assay cultures used in different experiments, so that normal neutrophilconditioned medium at a 10% dilution reduced colony formation by 20% to 80%. However, within individual experiments, such as that whose results are illustrated in Fig. 5, the findings were consistent. Different normal neutrophil-conditioned media had similar inhibitory effects (Fig. 5B) and, because the inhibitor in such media is stable when frozen, studies can be repeated when individual assay cultures are found not to have had the required sensitivity. Neutrophils release factors that inhibit the proliferation of colonyforming cells, but the mechanism is unknown. Theoretically the inhibition could be due to competition with colony-stimulating factor for receptor sites on the colony-forming cells, binding at separate receptor sites, or steric interaction with colony-stimulating factor. However, until we are able to obtain purified preparations of colony-stimulating factor and inhibitor, precise analysis of kinetic data is impossible. Furthermore, we have no idea how many active factors are released from neutrophils. Other cell products inhibit granulopoiesis in vitro. Factors of both high and low molecular weight appear to be involved. For example, Broxmeyer, Moore and Ralph5 have described colony inhibitory activity that diminishes the production of colonystimulating factor by mononuclear leukocytes but has no effect on the response of colony-forming cells to colony-stimulating factor. This activity has now been identified as a feature of lactoferrin.'2 This highmolecular-weight lipoprotein is contained in neutrophilic granules and is uniformly deficient in chronic myelogenous leukemia.. Leukemic blast cells inhibit granulopoiesis in cultures of cells from mice23 and humans.TM26 This is at least partly due to the release of humoral factors of low molecular weight (unpublished data) that reduce the proliferation of normal colony-forming cells, as do the inhibitors released
from normal neutrophils. The biologic released from normal neutrophils and significance of the various factors that released from leukemic blast active in cell cultures is unclear, and cells needs to be studied further. it is important that the specificity of It is too soon to regard chronic any inhibitor be established so that myelogenous leukemia as being chara factor of physiologic relevance can acterized by failure of negative feedbe distinguished from an in vitro back mechanisms, as has been sugartefact. gested.17 Certainly there is great variIn healthy individuals granulopoie- ability in the amount of inhibitor resis proceeds in the presence of inhi- leased by neutrophils in this disease, bitors that have activity in vitro. The and while the released inhibitor is effects of the inhibitors are concen- sometimes undetectable it is often tration-dependent; this is compatible excessive. Proliferation of leukemic with the concept that normal granu- colony-forming cells is inhibited by locytes provide negative feedback. normal and abnormal neutrophil-conResults of studies in patients add ditioned media,u but it is as yet credibility to this concept. In myelo- impossible to compare the relative proliferative and myelodysplastic dis- sensitivity of leukemic colony-formorders, variability in the amount of ing cells to inhibitor because the seninhibitor released from neutrophils sitivity of normal colony-forming cells could indicate variable impairment is so variable. of feedback control mechanisms in This study was supported by grant 309 individual patients, but other factors, from the Ontario Cancer Treatment such as recruitment of colony-form- and Research Foundation. ing cells from the pluripotential hematopoietic stem-cell compartment, and the capacity of colony-forming References cells to proliferate in response to 1. HASKILL JS, MCKNIGHT RD, GALBRAITH PR: Cell-cell interaction in colony-stimulating factor and to difvitro: studied by density separation of ferentiate, are undoubtedly of equal colony-forming, stimulating, and inor greater importance. Nevertheless, hibiting cells from human bone marrow. Blood 40: 394, 1972 production of inhibitor by neutrophils and blast cells in clonal marrow dis- 2. BAKER FL, BROXMEYER HE, GALBRAITH PR: Control of granulopoiesis orders could provide an explanation in man. III. Inhibition of colony forfor suppression of normal granulomation by dense leukocytes. J Cell Pliysiol 86: 337, 1975 poiesis, and might be a factor influencing, to some extent, the natural 3. BROXMEYER HE, BAKER FL, GALBRAITH PR: In vitro regulation of history of various diseases. Elaboragranulopoiesis in human leukemia: aption of inhibitor by leukemic blast plication of an assay for colony inhibiting cells. Blood 47: 389, 1976 cells may be an important mechanism by which leukemic cells inhibit 4. HASKILL JS, MCKNIGHT RD, GALBRAITH PR: Cell-to-cell interactions in the proliferation of normal hematothe regulation of granulopoiesis in poietic tissue, and might explain bone vitro, in in Vitro Culture of Hemopoletic Cells, VAN BEKKUM DW, DIcKE marrow failure in preleukemic states. KA (eds), Radiobiological Institute Clearly, all aspects of granulopoiesis TNO, Rijswijk, 1972, pp 307-18 accessible to study in vitro and in vivo 5. BROXMEYER HE, MOORE MAS, RALPH need to be correlated to establish P: Cell-free granulocyte colony inhibiting activity derived from human patterns of cellular interactions that polymorphonuclear neutrophils. Exp may be meaningful in the light of Hematol 5: 87, 1977 hematologic findings and the clinical 6. RYTOMAA T, VILI'o JA, LEX'ANTO A, course in individual patients. et al: Effect of granulocytic chalone on acute myeloid leukaemia in man. It remains to be determined if follow-up study. Lancet 1: 771, A assays for neutrophil and blast-cell 1977 inhibitor will be clinically useful. 7. MESSNER HA, TILL JE, MCCULLOCH For example, they might identify perEA: Interacting cell populations affecting granulopoietic colony formasons with leukemia who have "intion by normal and leukemic marrow hibitor deficiency" and could potencells. Blood 42: 701, 1973 tially benefit from chalone therapy.6 8. BAKER FL, GALBRMTH PR: Nutritional and regulatory roles of human serum Certainly the relation of inhibitor
552 CMA JOURNAL/MARCH 3, 1979/VOL. 120
(amoxicillin) AMOXIV (amoxicillin)... A new generation broad-spectrum penicillin. INDICATIONS: Infections due to susceptible strains of the following microorganisms: Gram-negative-H. influenzae, E. coil, R mirabilis and N.'gonorrhoeae. Gram-positive-Streptococci, D.pneumoniae and penicillin-sensitive staphylococci. In emergency cases where the causative organism is not yet identified, therapy may be initiated with AMOXIL on the basis of clinical judgment while awaiting the results of bacteriologic studies. DOSAGE AND ADMINISTRATION: Infections of the ear, nose and throat due to streptococci, pneumococci, and penicillin-sensitive staphylococci; infections of the upper respiratory tract due to H. influenzae; in fections of the genitourinary tract due to E. coli, R mirabilis, and S. faecalis; infections of the skin and soft tissues due to streptococci, penicillin-sensitive staphylococci and E. coli: Usual Dose: Adults-250 mg every 8 hours. Children-25 mg/kg/day in divided doses every 8 hours. This dosage should not exceed the recommended adult dosage. In severe infections or infections caused by less sensitive organisms: 500 mg every 8 hours for adults, and 50 mg/kg/day in divided doses every 8 hours for children. Infections of the lower respiratory tract due to streptococci, pneumococci, penicillin-sensitive staphylococci and H. influenzae: Usual Dose: Adults-500 mg every 8 hours. Children-SO mg/kg/day in divided doses every 8 hours. This dosage should not exceed the recommended adult dosage. Urethritis due to N. gonorrhoeae: 3 g as a single oral dose. Patients with gonorrhea, with a suspected lesion of syphilis, should have darkfield examinations before receiving AMOXIL, and monthly serologic tests for a minimum of four months. For chronic urinary tract infections, frequent bacteriologic and clinical appraisals are necessary. Smaller doses than those recommended above should not be used. Stubborn infections may require several weeks' treatment, sometimes at higher doses than recommended above. Concurrent bacteriolo. ic sensitivity monitoring is recommended. ontinued clinical and/or bacteriologic follow-up for several months after cessation of therapy may be required. Treatment should continue for 48 to 72 hours beyond the time patient becomes asymptomatic or bacterial eradication is obtained. At least 10 days' treatment is recommended for infections caused by beta-hemolytic streptococci to prevent acute rheumatic fever or glomerulonephritis. CONTRAINDICATION: In patients with a history of allergy to the penicillins or the cephalosporins. PRECAUTIONS: Periodic assessment of renal, hepatic, and hematopoletic function should be made during prolonged AMOXIL therapy. AMOXIL is excreted mostly by the kidney. The dosage administered to patients with renal impairment should be reduced proportionately to the degree of loss of renal function. The possibility of superinfections with mycotic or bacterial organisms should be kept in mind during therapy. If superinfections occur (usually involvin. Aerobacter, Pseudomonas or Cane drug should be discontinued and ap.ropriate therapy instituted. ADVERSE REACIONS: As with other penicillins, presumably the most common untoward reactions will be related to sensitivity phenomena, similar to those observed with ampicillin. They are more likely to occur in individuals who have previously demonstrated hypersensitivity to penicillins and in those with a history of allergy, asthma, hay fever or urticaria. (See Product Monograph which is available on request).SUPPLI ED: AMOXIL-250 Capsules (250 mg amoxicillin) in bottles of 100 and 500. AMOXIL-500 Capsules (500 mg amoxicillin) in bottles of 100. AMOXIL-125 Suspension(125mg amoxicillin per 5 ml) in bottles of 75, 100 and 150 ml. AMOXIL-250 Suspension (250 mg amoxicillin per 5 ml) in bottles of 75, 100 and 150 ml. AMOXIL Pediatric Drops (50 mg amoxicillin per ml) in bottles of 15 ml. AYERST LABORATORIES
Division of Ayerst, McKenna & Harrison Limited Montreal, Canada Made in Canada by arrangement with BEECHAM, INC. .Reg'd
in cultures of human granulopojetic cells. Blood 52: 241, 1978 9. GALBRAITH PR, MORLEY DC, BAKER FL: Inhibition of colony formation by neutrophils - probable dual mechanism (abstr), in 19th Annual Meeting of the American Society of Hematology, Boston, Dec 4-7, 1976, p 114 10. GALBRAITH PR, MORLEY DC, BAKER FL: Competitive inhibition of colony formation by neutrophil lysate (abstr).
Ann R Coll Physicians Surg Can 10: 59, 1977 11. SENN JS, MCCLJLLoCH EA: Radiation sensitivity of human bone marrow cells measured by a cell culture method. Blood 35: 56, 1970 12. GOLDE DW, CLINE MJ: Endotoxininduced release of colony-stimulating activity in man. Proc Soc Exp Biol Med 149: 845, 1975 13. WiNmonE MM: Clinical Hematology, 7th ed, Lea & Febiger, Philadelphia, 1974 14. GUSTAVSSON AL, OLOFSSON T, OLSSON I: A granulocyte low molecular weight inhibitor of granulopoiesis, in The
16th International Congress on Hema-
acute leukemic cells. Blood 50: 799, 1977 24. Moiuus TCM, MCNEILL TA, BRIDGES JM: Inhibition of normal human in vitro colony forming cells by cells from leukaemic patients. Br J Cancer 31: 641, 1975 25. CHIYODA S, Mizooucm H, KOSAKA K,
et al: Influence of leukaemic cells on the colony formation of human bone marrow cells in vitro. Ibid, p 355 26. cHIYODA S, MIzocucHI H, ASANO S, et al: Influence of leukaemic cells on the colony formation of human bone marrow cells in vitro. II. Suppressive effects of leukaemic cell extracts. Br
J Cancer 33: 379, 1976 27. BROXMEYER HE, MENDELSOHN N, MOORE MAS: Abnormal granulocyte feedback regulation of colony forming and colony stimulating activity-producing cells from patients with chronic myelogenous leukemia. Leukemia Res
1: 3, 1977 28. GALURAITH PR: Interactions between normal and leukemic leukocytes in culture (abstr), in 17th Congress of
the International Society of Hematology, Paris, July 27, 1978, p 922
tc'logy, Kyoto, Japan, SENO S, TAKAKU
F, IRINO S (eds), Sept 5-11, 1976, p 26 15. PARAN M, ICHIKAWA Y, SACHS L: Feedback inhibition of the development of macrophage and granulocyte colonies. II. Inhibition by granulocytes.
Proc Natl Acad Sci USA 62: 81, 1969 16. SHADDUCK RK: Granulocyte stimulating and inhibiting activity from neutrophils (PMN's): possible dual feedback control of granulopoiesis (abstr). Blood 38: 820, 1971 17. METCALF D, CHAN SH, GUNZ FW, et al: Colony-stimulating factor and inhibitor levels in acute granulocytic leukemia. Ibid, p 143 18. RYTOMAA T, KIvINIEMI K: Regulation
system of blood cell production, in Control of Cellular Growth in the Adult Organism, Sigrid Juselues Foundation Symposium, TEIR H, R.-
TOMAA T (eds), Aced Pr, New York, 1967, p 106 19. Idem: Control of granulocyte production. I. Chalone and antichalone, two specific humoral regulators. Cell Tissue Kinet 1: 329 1968 20. PAUKOVITS WR: Control of granulocyte production: separation and chemical identification of a specific inhibitor (chalone). Cell Tissue Kinet 4: 539, 1971 21. BAKER FL, GALBRAITH PR: Mechanism of neutrophil-induced inhibition of colony formation (abstr), in 18th Annual Meeting of the American Society of Hematology, Dallas, Texas, Dec 6-9, 1975, p 123 22. BROXMEYER HE, SMIThYMAN A, EGER
RR, et al: Identification of lactoferrin as the granulocyte-derived inhibitor of colony-stimulating activity production. J Exp Med 148: 1052, 1978 23. MILLER AM, PAGE PL, HARTWELL BL. et al: Inhibition of growth of normal murine granulocytes by cocultured
BOOKS continued from page 542 INTRODUCTION TO CLINICAL RADIOLOGY. A Correlative Approach to Diagnostic Imaging. Richard H. Daffner. 410 pp. lIlust. The C.V. Mosby Company, Saint Louis. Missouri, 1978. $17, paperbound. ISBN 0-8016-1203-9 THE LUNG. Structure, Function and Disease. International Academy of Pathology Monograph. Edited by William M. Thurlbeck and Murray R. Abell. 322 pp. lIlust. The Williams & Wilkins Company, Baltimore, Maryland; Burns & MacEachern Limited, Don Mills, Ont., 1978. $41.95. ISBN 0.683-08274-4 MANUAL OF DERMATOLOGIC THERAPEUTICS. With Essentials of Diagnosis. 2nd ed. Kenneth A. Arndt. 377 pp. IlIust. Little, Brown and Company (Inc.), Boston, Massachusetts, 1978. $10.95, spiralbound. ISBN 0-316-05280-9 MANUAL OF SURGICAL THERAPEUTICS. 4th ed. Edited by Robert E. Condon and Lloyd M. Nyhus. 511 pp. IlIust. Little, Brown and Company (Inc.), Boston, Massachusetts, 1978. $10.95, spiralbound. ISBN 0-316-15286-2 MEDICAL MALPRACTICE LAW. 2nd ed. Angela Roddey Holder. 562 pp. John Wiley & Sons, Inc., New York; John Wiley & Sons Canada, Limited, Rexdale, Ont., 1978. Price not stated. ISBN 0-471-03882-2 MEDICAL WISDOM AND ETHICS IN THE TREATMENT OF SEVERELY DEFECTIVE NEWBORN AND YOUNG CHILDREN. Proceedings of a Symposium held on November 18, 1976. Organized by the Center for Bioethics, Clinical Research Institute of Montreal, Montreal, Quebec, Canada. Edited by David J. Roy. 99 pp. Illust. Eden Press Inc., Montreal, 1978. Price not stated. ISBN 0-88831-035-0
continued on page 563
CMA JOURNAL/MARCH 3, 1979/VOL. 120 555
The concept that polymorphonuclear leukocytes, or neutrophils, play a role in feedback control of granulopolesis has been supported by the finding in bone marrow culture studies that mature neutrophils inhibited formation of granulocytic colonies. The study described in this paper was done to investigate the mechanisms involved. With the use of a modified assay it was found that mature neutrophils released factors that reduced the proliferation of colony-forming cells in cultures stimulated by cell-free colony-stimulating factor. In myeloproliferative and myelodysplastic disorders the amount of inhibitor released by the neutrophils varied greatly. Leukemic blast cells also released inhibitor, and in some cases the amount released per cell was greater than tha amount released from normal mature neutrophils. The inhibitory factors released from the neutrophils differed from those previously described in the literature in terms of mode of action and apparent molecular size. La notion voulant que les leucocytes polymorphonucleaires, ou neutrophiles, jouent un r6le dans le contr6le par retroaction de Ia granulopolese s'est vu soutenue par l'observation dans les 6tudes sur Ia culture des cellules de moelles osseuses que les neutrophiles a maturite inhibaient Ia formation des cellules granulocytaires. L'etude qui fait l'objet de cette publication a et6 falte dans le but d'investiguer les m6canismes impliques. A l'aide d'une technique modifiee on a trouve que les neutrophiles a maturite liberent des facteurs aul r6duisent Ia proliferation des cellules formant des colonies dans des cultures stimulees par un facteur libre de cellules qui favorise Ia formation de colonies. Dans les maladies my6loproliferatives et les myelodysplasies les quantites d'inhibiteur liber6es par les neutrophiles ont vari6 consid6rablement. Les cellules leucemiques blastiques liberent aussi de cet From the department of medicine, Queen's University, Kingston *Profe.or of medicine tSenior technician, division of hematology .Research associate Reprint requests to: Dr. Peter R. Gaibraith, 102 Stuart St., Ste. 113, Kingston, Ont. K7L 3N6
inhibiteur, et dans certains cas Ia quantite lib6r6e par cellule surpassait Ia quantit6 lib6r6e par des neutrophiles normaux a maturit6. Les facteurs inhibiteurs lib6res par les neutrophiles differalent de ceux d6jA d6crits dans Ia litterature par leur mode d'action et leur poids moleculaire apparent.
End-product inhibition is an important regulatory mechanism in many biologic systems. The concept that polymorphonuclear leukocytes, or neutrophils, exert feedback control over granulopoiesis has been suggested by the results of in vitro studies that indicate that neutrophils inhibit the formation of granulocytic colonies.'4 Such studies may have clinical relevance since some leukemic neutrophils appear not to inhibit,34 and remission of acute leukemia has occurred following treatment with granulocytic chalone.6 In this paper we describe our study that showed that normal neutrophils released factors that inhibited the proliferation of granulocyte-committed colony-forming cells in cultures stimulated by cell-free colonystimulating factor. These factors differed from those previously described in molecular size and site of action. Furthermore, the amount of inhibitor released by leukemic neutrophils and blast cells varied greatly. Methods Collection of cells Venous blood samples and bone marrow aspirates were collected from informed healthy volunteers and patients with various hematologic disorders, as described previously,2 in tubes containing 0.5 mL of citratephosphate-dextrose solution USP (Fenwal Laboratories, division of Baxter Travenol Laboratories of Canada Ltd., Malton, Ont.) as an anticoagulant and 3% dextran to accelerate the sedimentation of erythrocytes. The nucleated-cell-rich suspensions were washed three times in
CMRL 1066 tissue culture medium (Grand Island [New York] Biological Company) and then subjected to cell separations based on cell density in Ficoll® (density 1.070 g/mL; dissolved in CMRL 1066 tissue culture medium) (Pharmacia Fine Chemicals, Uppsala, Sweden) and adhesiveness to plastic.3'7 The following fractions were collected: 1. The light-density mononuclear leukocyte fraction of normal blood 3,8 leukucyte suspensions. This pro vided a mononuclear leukocyte suspension containing monocytes, which was used in the preparation of colony-stimulating factor. 2. The high-density fraction of blood leukocytes.' This yielded cell suspensions highly enriched with neutrophils. 3. The nonadherent light-density nucleated cell fraction of normal human bone marrow cells,8 to serve as the source of target colony-forming cells. These were used in all the experiments described. Preparation of colony-stimulating factor Mononuclear-leukocyte-conditioned medium or serum provided cellfree colony-stimulating factor for all experiments. They were prepared respectively by incubating 2 x 10. normal blood mononuclear leukocytes per millilitre at 370C for 5 to 7 days in tissue culture medium containing 30% normal human serum heated at 580C for 150 minutes8 or in 100% neutropenic serum obtained from patients with acute myelomonocytic leukemia and severe neutropenia (neutrophil count less than 0.6 x 109/L). Comparable results were obtained with the two preparations. Neutropenic serum was used initially to minimize contamination by inhibitor released from neutrophils; inhibitory activity is destroyed by heat treatment of normal human serum.8
CMA JOURNAL/MARCH 3, 1979/VOL. 120 545
The two preparations were assayed batch of colony-stimulating factor. to determine the least concentration The assay cultures were virtually dethat promoted maximum colony for- pleted of monocytes7 so as to prevent mation. This concentration was then endogenous elaboration of colonyused in all the experiments described. stimulating factor. The reduced content of neutrophils"3 made the assay Preparation ci inhibitor cultures more sensitive to both Inhibitor from normal blood or colony-stimulating factor and inhibibone marrow cell suspensions was tors.9'10 prepared by incubating 2 x 106 neuIn all experiments aliquots of trophils per millilitre in CMRL 1066 CMRL 1066 tissue culture medium tissue culture medium for 10 minutes, conditioned by specified leukocyte and then removing the cells by Milli- populations were assayed at a 10% pore filtration (filter pore size 0.45 dilution tor their inhibition of colony p.m). The filtrate, termed neutrophil- formation in cultures stimulated by conditioned medium, was frozen and cell-free colony-stimulating factor. In stored at -25 0C. Initially the neutro- patient studies a dilution curve for phils were incubated at 370C, but neutrophil-conditioned medium presubsequently it was found that incu- pared from normal blood was used bation at room temperature (22 0C) to establish the validity of the assay yielded similar results and was much - that is, that the inhibitory effect easier. However, in individual expe- of this medium was concentrationriments the temperature at which the dependent - and the potency of a conditioned medium was prepared 10% dilution of this medium was was kept constant and is specified. assessed simultaneously with that of Inhibitor was prepared from leu- a 10% dilution of neutrophil-condikemic blood or bone marrow cell tioned medium prepared from leususpensions as described above. kemic blood. When the suspensions contained less Since the leukemic-blast-cell-conthan 90% blast cells or more than 5% neutrophils, density separation in Ficoll® was used to enrich for 40blast cells and remove neutrophils. Cell cultures The single-layer semisolid agar culture system was used throughout the study.1'3 Each 1-mL culture contained 0.3% agar to minimize dispersal of colonies. The culture medium, described previously,2'3 was modified to contain 30% normal human serum heated at 580C for 150 minutes instead of fetal calf serum, so as to provide appropriate supplementation with "essential serum growth factors" (or permissive factors), with retention of sensitivity to the action of both the stimulatory and the inhibitory molecules under investigation.8 Experimental design All agar cultures contained 0.75 to 1 x 1 0.' nonadherent light-density marrow cells. To provide a constant source of colony-forming cells in each experiment, we used marrow from one healthy individual and a single
ditioned media were not all studied simultaneously, the results were normalized to show the relative inhibition of colony formation by the two types of conditioned medium. Colony counts Colonies were counted on triplicate plates after 7 days of incubation. In control cultures aggregates of 20 or more cells were considered as colonies, partly on the basis of precedent11'13 and partly on the basis of the results (unpublished) of our preliminary studies, which showed that, in cultures stimulated by increasing concentrations of colony-stimulating factor, when a single plate was removed daily from the incubator and scored for aggregate number and aggregate size, an increase was found in aggregate number with time that was dependent on the concentration of colony-stimulating factor. Aggregates of only 3 to 5 cells appeared on day 2, reached a maximum number on day 3 or 4, and decreased exponentially thereafter. Colonies of 20 or more cells appeared on days 6, 7
No incubation '. with neutrophils 30
Colonies per 20 plate (mean ± SEM)
4
I0-
0 5
10
30
60
Time (minutes)
120
FIG. 1-Release of inhibitor by 2 X 10 neutrophils per millilitre incubated in CMRL 1066 tissue culture medium as function of time of incubation. At end of incubation cell suspensions underwent Millipore filtration (filter pore size 0.45 .m) and 0.1 mL of cefl-free filtrate was added to cultures of nonadherent light-density nucleated cell fractions of normal bone marrow cells. AU cultures were stimulated by 5% mononuclear-leukocyte-conditioned serum, which provided cell-free colony-stimulating factor.
546 CMA JOURNAL/MARCH 3, 1979/VOL. 120
and 8, and thereafter decreased in dilution. The higher than predicted Stability of inhibitor in neutrophilnumber but increased in size. More activity of inhibitor in the dialysis conditioned medium than 95% of colonies present on day fluid suggests that the dialysis memStability was assessed by assaying 7 were peroxidase-positive granulo- brane separated inhibitor from larger the activity of neutrophil-conditioned cytic clones. In cultures with added molecules that would have influenced medium after incubation at 370C inhibitor, colony size was reduced the stability or action of the lower- (Fig. 4). Little activity was detected by approximately 50%. Therefore, molecular-weight inhibitor (Fig. 3). after 72 hours. Inhibitory activity was aggregates of fewer than 20 cells were considered as colonies. This, in effect, biases our data against the hypotheses being tested. I No inhibitor 200Results Kinetics of inhibitor released ISOfrom neutrophils The rate of inhibitor release from Colonies ioo. normal neutrophils was investigated per by assaying aliquots of 10% neutroplate 140 phil-conditioned medium harvested at intervals after initiation of cul- (mean ± SEM) ture. Assuming minimal cell damage 120. during Millipore filtration, the results shown in Fig. 1 indicate that inhibI00. itor was rapidly released, and that inhibitory activity in the medium was maximal within 5 minutes.
Sensitivity of assays Individual experiments varied with respect to the sensitivity of assay cultures to the addition of normal neutrophil-conditioned medium. In all assays inhibition was linearly related to the dilution of the medium. In the most sensitive assays a biphasic inhibitor dilution curve was obtained (Fig. 2): at dilutions of 1:4096 to 1:1024 marked inhibition occurred, and at lower dilutions the slope of the curve decreased. Clearly the assay is relatively insensitive to small differences in inhibitor concentration, but larger differences can be appreciated.
I
4096
024
I
256
64
16
4
Dilution of neutrophil.-conditioned medium FIG. 2-Concentration-dependent effects of inhibitor in neutrophil-conditioned medium diluted with CMRL 1066 tissue culture medium and added (0.1 mL per 1-mL culture) to cultures stimulated by 2% mononuclear-leukocyte-conditioned medium.
Control 120 Neutrophi 1-conditioned medium . 100 .4
Undialysed 10% 0.1%
Dialysed 10%
Dialysis fluid 10%
(0
2 80 0) (0 ..
Effect of dialysis on inhibitory activity We dialysed 10 mL of neutrophilconditioned medium harvested at 10 minutes against 1 L of tissue culture medium for 24 hours at 40C. The inhibitor moved freely through the dialysis membrane (Fisher Scientific Co. Ltd., Ottawa, cat, no. 5-667E; pores admit molecules of molecular weight less than 12 000 daltons). The action of inhibitor in the dialysate was that predicted on the basis of
I
16346
60
0) 0. Si) ..
(-)0
40
rI-i
ri-i
FIG. 3-Effect of dialysis on inhibitory activity. After 10 X 10 neutrophils per miflilitre were incubated in CMRL 1066 tissue culture medium for 10 minutes the cell-free conditioned medium was dialysed for 24 hours at 4C (10 mL of conditioned medium against 1000 mL of tissue culture medium) and assayed at a 10% dilution for inhibition of cultures stimulated by 5% mononuclear-leukocyte-conditioned serum. CMA JOURNAL/MARCH 3, 1979/VOL 120 549
retained in neutrophil-conditioned medium stored at -250C for as long as 90 days. Release of inhibitor from abnormal neutrophils The patients selected for study had unequivocal diagnoses established by standard criteria.13 The density separation in Ficoll® ensured that adequate numbers of neutrophils were obtained for study in all patients. Normal neutrophil-conditioned medium was diluted with CMRL 1066 tissue culture medium so as to obtain a control concentration curve to establish the validity of the assay (Fig. 5A). The control and patients' conditioned media were simultaneously assayed at a single concentration (10%). The activity of inhibitors released from control neutrophils was similar, while that of inhibitors released from neutrophils of patients with a variety of hematopoietic stemcell disorders was variable, even within single diagnostic categories (Fig. 5B). Thirteen different leukemic-blastcell-conditioned media were assayed for inhibitory activity at a single concentration (10%). Because normal neutrophil-conditioned medium in various assays reduced colony formation by 20% to 80%, normalized results are presented in Fig. 6. These show that 11 of the 13 media inhibited colony formation. Inhibitor was released to the greatest extent from blast cells obtained from patients whose bone marrow cultures did not form colonies (aggregates of 20 or more cells). In these cases the blast cell medium was as active or more active than normal neutrophilconditioned medium assayed simultaneously. In one such case the blast cell medium was active at a dilution higher than 1:4096, whereas activity was lost in normal neutrophil-conditioned medium diluted to 1:64 (Fig. 7). Discussion Neutrophils release inhibitors with a variety of reported activities.1"'5''1420 The present study is the first to show that low-molecular-weight factors released from normal neutrophils in-
j
40
LU
No inhibitor
LI')
4'
.0 120 0
E
a, BOO
4-) 0 0.
L 80
0) 0. U'
0 0 0 C.)
60 40
-
I Days
2
4
incubation of inhibitor
FIG. 4-Stability of inhibitory activity of neutrophil-conditioned medium. Aliquota of medium were incubated at 37C for various times, then frozen at -20C, and subsequently diluted 1:4 and added in 0.1-mL portions to cultures of nonadherent light-density nucleated cell fractions of bone marrow cells stimulated by 5% mononuclear-leukocyte-conditioned serum.
A.60
Colonies per plate (mean * SEM)
0
/64
.32
"'Is
'8
/4
1/2
Dilution of neutrophil-conditioned medium Neutrophi 1-conditioned medium
B
60-
None
Normal
PV
CML
AML
AMML
Miscellaneous
50Multiple myeloma
Inhibition 40of colony 30formation (mean * SEM)
6 p-thalassemia minor Q Hodgkins disease
. PR
20-
I
I0-
.PR 6Rem 6u U Id
A 1PR
Aplastic anemia
PNH
. Refractory sideroblastic anemia . Refractory anemia Preleukemia
-t
FIG. 5-Effect on colony formation of 0.1 mL of neutrophil-conditioned medium prepared from 2 X 10. neutrophils per millilitre incubated at 22C. In a representative experiment normal conditioned medium was serially diluted to show the effect of different concentrations of inhibitor (panel A). Panel B shows results when media of controls and patients were assayed simultaneously. Results are expressed as the mean reduction (± SEM) in colony number. All cultures were stimulated by 2% mononuclear-leukocyte-conditioned medium. PV = polycythemia vera; CML = chromc myelogenous leukemia; AML = acute myelogenous leukemia; AMML = acute myelomonocytic leukemia; PNH paroxysmal nocturnal hemoglobinuria; PR = partial remission; Rem. = remission; u = untreated.
550 CMA JOURNAL/MARCH 3, 1979/VOL. 120
Colony-formers Neut.
Blast
Cluster-formers Neut.
Blast
No growth Neut.
Blast
'0 0
.0 0 0
0.
______________
been partially resolved, there is variability in the sensitivity of the assay cultures used in different experiments, so that normal neutrophilconditioned medium at a 10% dilution reduced colony formation by 20% to 80%. However, within individual experiments, such as that whose results are illustrated in Fig. 5, the findings were consistent. Different normal neutrophil-conditioned media had similar inhibitory effects (Fig. 5B) and, because the inhibitor in such media is stable when frozen, studies can be repeated when individual assay cultures are found not to have had the required sensitivity. Neutrophils release factors that inhibit the proliferation of colonyforming cells, but the mechanism is unknown. Theoretically the inhibition could be due to competition with colony-stimulating factor for receptor sites on the colony-forming cells, binding at separate receptor sites, or steric interaction with colony-stimulating factor. However, until we are able to obtain purified preparations of colony-stimulating factor and inhibitor, precise analysis of kinetic data is impossible. Furthermore, we have no idea how many active factors are released from neutrophils. Other cell products inhibit granulopoiesis in vitro. Factors of both high and low molecular weight appear to be involved. For example, Broxmeyer, Moore and Ralph5 have described colony inhibitory activity that diminishes the production of colonystimulating factor by mononuclear leukocytes but has no effect on the response of colony-forming cells to colony-stimulating factor. This activity has now been identified as a feature of lactoferrin.'2 This highmolecular-weight lipoprotein is contained in neutrophilic granules and is uniformly deficient in chronic myelogenous leukemia.. Leukemic blast cells inhibit granulopoiesis in cultures of cells from mice23 and humans.TM26 This is at least partly due to the release of humoral factors of low molecular weight (unpublished data) that reduce the proliferation of normal colony-forming cells, as do the inhibitors released
from normal neutrophils. The biologic released from normal neutrophils and significance of the various factors that released from leukemic blast active in cell cultures is unclear, and cells needs to be studied further. it is important that the specificity of It is too soon to regard chronic any inhibitor be established so that myelogenous leukemia as being chara factor of physiologic relevance can acterized by failure of negative feedbe distinguished from an in vitro back mechanisms, as has been sugartefact. gested.17 Certainly there is great variIn healthy individuals granulopoie- ability in the amount of inhibitor resis proceeds in the presence of inhi- leased by neutrophils in this disease, bitors that have activity in vitro. The and while the released inhibitor is effects of the inhibitors are concen- sometimes undetectable it is often tration-dependent; this is compatible excessive. Proliferation of leukemic with the concept that normal granu- colony-forming cells is inhibited by locytes provide negative feedback. normal and abnormal neutrophil-conResults of studies in patients add ditioned media,u but it is as yet credibility to this concept. In myelo- impossible to compare the relative proliferative and myelodysplastic dis- sensitivity of leukemic colony-formorders, variability in the amount of ing cells to inhibitor because the seninhibitor released from neutrophils sitivity of normal colony-forming cells could indicate variable impairment is so variable. of feedback control mechanisms in This study was supported by grant 309 individual patients, but other factors, from the Ontario Cancer Treatment such as recruitment of colony-form- and Research Foundation. ing cells from the pluripotential hematopoietic stem-cell compartment, and the capacity of colony-forming References cells to proliferate in response to 1. HASKILL JS, MCKNIGHT RD, GALBRAITH PR: Cell-cell interaction in colony-stimulating factor and to difvitro: studied by density separation of ferentiate, are undoubtedly of equal colony-forming, stimulating, and inor greater importance. Nevertheless, hibiting cells from human bone marrow. Blood 40: 394, 1972 production of inhibitor by neutrophils and blast cells in clonal marrow dis- 2. BAKER FL, BROXMEYER HE, GALBRAITH PR: Control of granulopoiesis orders could provide an explanation in man. III. Inhibition of colony forfor suppression of normal granulomation by dense leukocytes. J Cell Pliysiol 86: 337, 1975 poiesis, and might be a factor influencing, to some extent, the natural 3. BROXMEYER HE, BAKER FL, GALBRAITH PR: In vitro regulation of history of various diseases. Elaboragranulopoiesis in human leukemia: aption of inhibitor by leukemic blast plication of an assay for colony inhibiting cells. Blood 47: 389, 1976 cells may be an important mechanism by which leukemic cells inhibit 4. HASKILL JS, MCKNIGHT RD, GALBRAITH PR: Cell-to-cell interactions in the proliferation of normal hematothe regulation of granulopoiesis in poietic tissue, and might explain bone vitro, in in Vitro Culture of Hemopoletic Cells, VAN BEKKUM DW, DIcKE marrow failure in preleukemic states. KA (eds), Radiobiological Institute Clearly, all aspects of granulopoiesis TNO, Rijswijk, 1972, pp 307-18 accessible to study in vitro and in vivo 5. BROXMEYER HE, MOORE MAS, RALPH need to be correlated to establish P: Cell-free granulocyte colony inhibiting activity derived from human patterns of cellular interactions that polymorphonuclear neutrophils. Exp may be meaningful in the light of Hematol 5: 87, 1977 hematologic findings and the clinical 6. RYTOMAA T, VILI'o JA, LEX'ANTO A, course in individual patients. et al: Effect of granulocytic chalone on acute myeloid leukaemia in man. It remains to be determined if follow-up study. Lancet 1: 771, A assays for neutrophil and blast-cell 1977 inhibitor will be clinically useful. 7. MESSNER HA, TILL JE, MCCULLOCH For example, they might identify perEA: Interacting cell populations affecting granulopoietic colony formasons with leukemia who have "intion by normal and leukemic marrow hibitor deficiency" and could potencells. Blood 42: 701, 1973 tially benefit from chalone therapy.6 8. BAKER FL, GALBRMTH PR: Nutritional and regulatory roles of human serum Certainly the relation of inhibitor
552 CMA JOURNAL/MARCH 3, 1979/VOL. 120
(amoxicillin) AMOXIV (amoxicillin)... A new generation broad-spectrum penicillin. INDICATIONS: Infections due to susceptible strains of the following microorganisms: Gram-negative-H. influenzae, E. coil, R mirabilis and N.'gonorrhoeae. Gram-positive-Streptococci, D.pneumoniae and penicillin-sensitive staphylococci. In emergency cases where the causative organism is not yet identified, therapy may be initiated with AMOXIL on the basis of clinical judgment while awaiting the results of bacteriologic studies. DOSAGE AND ADMINISTRATION: Infections of the ear, nose and throat due to streptococci, pneumococci, and penicillin-sensitive staphylococci; infections of the upper respiratory tract due to H. influenzae; in fections of the genitourinary tract due to E. coli, R mirabilis, and S. faecalis; infections of the skin and soft tissues due to streptococci, penicillin-sensitive staphylococci and E. coli: Usual Dose: Adults-250 mg every 8 hours. Children-25 mg/kg/day in divided doses every 8 hours. This dosage should not exceed the recommended adult dosage. In severe infections or infections caused by less sensitive organisms: 500 mg every 8 hours for adults, and 50 mg/kg/day in divided doses every 8 hours for children. Infections of the lower respiratory tract due to streptococci, pneumococci, penicillin-sensitive staphylococci and H. influenzae: Usual Dose: Adults-500 mg every 8 hours. Children-SO mg/kg/day in divided doses every 8 hours. This dosage should not exceed the recommended adult dosage. Urethritis due to N. gonorrhoeae: 3 g as a single oral dose. Patients with gonorrhea, with a suspected lesion of syphilis, should have darkfield examinations before receiving AMOXIL, and monthly serologic tests for a minimum of four months. For chronic urinary tract infections, frequent bacteriologic and clinical appraisals are necessary. Smaller doses than those recommended above should not be used. Stubborn infections may require several weeks' treatment, sometimes at higher doses than recommended above. Concurrent bacteriolo. ic sensitivity monitoring is recommended. ontinued clinical and/or bacteriologic follow-up for several months after cessation of therapy may be required. Treatment should continue for 48 to 72 hours beyond the time patient becomes asymptomatic or bacterial eradication is obtained. At least 10 days' treatment is recommended for infections caused by beta-hemolytic streptococci to prevent acute rheumatic fever or glomerulonephritis. CONTRAINDICATION: In patients with a history of allergy to the penicillins or the cephalosporins. PRECAUTIONS: Periodic assessment of renal, hepatic, and hematopoletic function should be made during prolonged AMOXIL therapy. AMOXIL is excreted mostly by the kidney. The dosage administered to patients with renal impairment should be reduced proportionately to the degree of loss of renal function. The possibility of superinfections with mycotic or bacterial organisms should be kept in mind during therapy. If superinfections occur (usually involvin. Aerobacter, Pseudomonas or Cane drug should be discontinued and ap.ropriate therapy instituted. ADVERSE REACIONS: As with other penicillins, presumably the most common untoward reactions will be related to sensitivity phenomena, similar to those observed with ampicillin. They are more likely to occur in individuals who have previously demonstrated hypersensitivity to penicillins and in those with a history of allergy, asthma, hay fever or urticaria. (See Product Monograph which is available on request).SUPPLI ED: AMOXIL-250 Capsules (250 mg amoxicillin) in bottles of 100 and 500. AMOXIL-500 Capsules (500 mg amoxicillin) in bottles of 100. AMOXIL-125 Suspension(125mg amoxicillin per 5 ml) in bottles of 75, 100 and 150 ml. AMOXIL-250 Suspension (250 mg amoxicillin per 5 ml) in bottles of 75, 100 and 150 ml. AMOXIL Pediatric Drops (50 mg amoxicillin per ml) in bottles of 15 ml. AYERST LABORATORIES
Division of Ayerst, McKenna & Harrison Limited Montreal, Canada Made in Canada by arrangement with BEECHAM, INC. .Reg'd
in cultures of human granulopojetic cells. Blood 52: 241, 1978 9. GALBRAITH PR, MORLEY DC, BAKER FL: Inhibition of colony formation by neutrophils - probable dual mechanism (abstr), in 19th Annual Meeting of the American Society of Hematology, Boston, Dec 4-7, 1976, p 114 10. GALBRAITH PR, MORLEY DC, BAKER FL: Competitive inhibition of colony formation by neutrophil lysate (abstr).
Ann R Coll Physicians Surg Can 10: 59, 1977 11. SENN JS, MCCLJLLoCH EA: Radiation sensitivity of human bone marrow cells measured by a cell culture method. Blood 35: 56, 1970 12. GOLDE DW, CLINE MJ: Endotoxininduced release of colony-stimulating activity in man. Proc Soc Exp Biol Med 149: 845, 1975 13. WiNmonE MM: Clinical Hematology, 7th ed, Lea & Febiger, Philadelphia, 1974 14. GUSTAVSSON AL, OLOFSSON T, OLSSON I: A granulocyte low molecular weight inhibitor of granulopoiesis, in The
16th International Congress on Hema-
acute leukemic cells. Blood 50: 799, 1977 24. Moiuus TCM, MCNEILL TA, BRIDGES JM: Inhibition of normal human in vitro colony forming cells by cells from leukaemic patients. Br J Cancer 31: 641, 1975 25. CHIYODA S, Mizooucm H, KOSAKA K,
et al: Influence of leukaemic cells on the colony formation of human bone marrow cells in vitro. Ibid, p 355 26. cHIYODA S, MIzocucHI H, ASANO S, et al: Influence of leukaemic cells on the colony formation of human bone marrow cells in vitro. II. Suppressive effects of leukaemic cell extracts. Br
J Cancer 33: 379, 1976 27. BROXMEYER HE, MENDELSOHN N, MOORE MAS: Abnormal granulocyte feedback regulation of colony forming and colony stimulating activity-producing cells from patients with chronic myelogenous leukemia. Leukemia Res
1: 3, 1977 28. GALURAITH PR: Interactions between normal and leukemic leukocytes in culture (abstr), in 17th Congress of
the International Society of Hematology, Paris, July 27, 1978, p 922
tc'logy, Kyoto, Japan, SENO S, TAKAKU
F, IRINO S (eds), Sept 5-11, 1976, p 26 15. PARAN M, ICHIKAWA Y, SACHS L: Feedback inhibition of the development of macrophage and granulocyte colonies. II. Inhibition by granulocytes.
Proc Natl Acad Sci USA 62: 81, 1969 16. SHADDUCK RK: Granulocyte stimulating and inhibiting activity from neutrophils (PMN's): possible dual feedback control of granulopoiesis (abstr). Blood 38: 820, 1971 17. METCALF D, CHAN SH, GUNZ FW, et al: Colony-stimulating factor and inhibitor levels in acute granulocytic leukemia. Ibid, p 143 18. RYTOMAA T, KIvINIEMI K: Regulation
system of blood cell production, in Control of Cellular Growth in the Adult Organism, Sigrid Juselues Foundation Symposium, TEIR H, R.-
TOMAA T (eds), Aced Pr, New York, 1967, p 106 19. Idem: Control of granulocyte production. I. Chalone and antichalone, two specific humoral regulators. Cell Tissue Kinet 1: 329 1968 20. PAUKOVITS WR: Control of granulocyte production: separation and chemical identification of a specific inhibitor (chalone). Cell Tissue Kinet 4: 539, 1971 21. BAKER FL, GALBRAITH PR: Mechanism of neutrophil-induced inhibition of colony formation (abstr), in 18th Annual Meeting of the American Society of Hematology, Dallas, Texas, Dec 6-9, 1975, p 123 22. BROXMEYER HE, SMIThYMAN A, EGER
RR, et al: Identification of lactoferrin as the granulocyte-derived inhibitor of colony-stimulating activity production. J Exp Med 148: 1052, 1978 23. MILLER AM, PAGE PL, HARTWELL BL. et al: Inhibition of growth of normal murine granulocytes by cocultured
BOOKS continued from page 542 INTRODUCTION TO CLINICAL RADIOLOGY. A Correlative Approach to Diagnostic Imaging. Richard H. Daffner. 410 pp. lIlust. The C.V. Mosby Company, Saint Louis. Missouri, 1978. $17, paperbound. ISBN 0-8016-1203-9 THE LUNG. Structure, Function and Disease. International Academy of Pathology Monograph. Edited by William M. Thurlbeck and Murray R. Abell. 322 pp. lIlust. The Williams & Wilkins Company, Baltimore, Maryland; Burns & MacEachern Limited, Don Mills, Ont., 1978. $41.95. ISBN 0.683-08274-4 MANUAL OF DERMATOLOGIC THERAPEUTICS. With Essentials of Diagnosis. 2nd ed. Kenneth A. Arndt. 377 pp. IlIust. Little, Brown and Company (Inc.), Boston, Massachusetts, 1978. $10.95, spiralbound. ISBN 0-316-05280-9 MANUAL OF SURGICAL THERAPEUTICS. 4th ed. Edited by Robert E. Condon and Lloyd M. Nyhus. 511 pp. IlIust. Little, Brown and Company (Inc.), Boston, Massachusetts, 1978. $10.95, spiralbound. ISBN 0-316-15286-2 MEDICAL MALPRACTICE LAW. 2nd ed. Angela Roddey Holder. 562 pp. John Wiley & Sons, Inc., New York; John Wiley & Sons Canada, Limited, Rexdale, Ont., 1978. Price not stated. ISBN 0-471-03882-2 MEDICAL WISDOM AND ETHICS IN THE TREATMENT OF SEVERELY DEFECTIVE NEWBORN AND YOUNG CHILDREN. Proceedings of a Symposium held on November 18, 1976. Organized by the Center for Bioethics, Clinical Research Institute of Montreal, Montreal, Quebec, Canada. Edited by David J. Roy. 99 pp. Illust. Eden Press Inc., Montreal, 1978. Price not stated. ISBN 0-88831-035-0
continued on page 563
CMA JOURNAL/MARCH 3, 1979/VOL. 120 555
