Mechanisms of glucocorticosteroid activity in patients with the preleukemic syndrome (hemopoietic dysplasia)

Lt.ukmfla Research Vol. 4. No. 6, pp. 571 to 580 0 Pergamon Press Ltd 1980. Printed in Great Britain

MECHANISMS IN PATIENTS

0145-2126/80/1201-0571S02.00,,'0

OF GLUCOCORTICOSTEROID ACTIVITY WITH THE PRELEUKEMIC SYNDROME

(HEMOPOIETIC

DYSPLASIA)*

GROVER C. BAGBY, JR/f V.A. Medical Center and The Edwin E. Osgood Memorial Center for Leukemia, University of Oregon Health Sciences Center, Portland, Oregon, U.S.A.

(Received 13 February 1980. Revised 30 June 1980. Accepted 4 July 1980) Almract--We have analyzed mechanisms of glucocorticosteroid activity in patients with the preleukemic syndrome by culturing in agar and methylcellulose bone marrow cells from 5 glucocorticosteroid-responsive patients with the preleukemic syndrome. We have found that methylpreclnisoione and cortisol are equally effective in enhancing colony growth in these patients, that enhancement occurs in either methylcellulose or agar matrix and in RPMI 1640, McCoy's 5a, and alpha medium. In 4 patient& glucocorticosteroids inhibited the activity of T-lymphocyte inhibitors of granulopoimis in vitro. In one patient, these agents stimulated the immediate proliferation of CFU-C and in the presence of LCM enhanced CFU-C survival in suspension culture. These effects were blocked by progesterone and not due to enhancement of CSA production or inhibition of inhibitory T-cell function. Our results document the involvement of cortisol sensitive T-lymphocytes in the suppression of granulopoiesis in a small number of patients with the preleukemic syndrome and indicate that the mechanisms of glucocorticosteroid responses among patients with this syndrome are heterogeneous.

Key words: Glucocorticosteroids, prelenkemie syndrome, T-lymphocytes, CFU-C.

INTRODUCTION A pRosr~-cnw multimodality study of patients with the preleukemic syndrome at our institutions have shown that of those patients followed for more than 2 years 76~ have died and 65~o have developed acute leukemia 1"4]. In addition, 13~ of the entire group died of infection or hemorrhage without having developed overt leukemia. Thus, two important therapeutic objectives in patients with this syndrome are to forestall the development of acute leukemia and to reverse cytopenias. While the former objective has not been achieved, we have found that prednisone therapy can achieve reversal of cytopenias in 10~ of patients with this disorder l4]. Furthermore, a series of studies in our laboratories and clinics have suggested that prednisone-responsive patients with the preleukemic syndrome 1"4] and other bone marrow failure states 12, 3] may be identified using in vitro techniques. The apparent correlation of in vitro with in vioo responses has enabled us to identify mechanisms of glueocortieosteroid action in prednisone-responsive patients with neutropenia associated with rheumatic diseases 12] and aplastic anemia 1,3]. In these studies we found that cortisol-sensitive T-lymphoeytes suppressed granulopoiesis in vitro and probably in vioo. The effect of glucoeorticosteroid therapy in these patients reflected a functional inhibition of these inhibitory lymphocytes by steroid. We report Abbreviations: CSA, Colony stimulating activity; LCM, Leukocyte conditioned medium; CFU-C, Granulocyte/macrophage colony forming units; T-cells, Thymus dependent lymphocytes; LDBMC, Light density bone marrow cells; ARA-C, Cytosine arabinoside; SRBC, Sheep red blood cells. Corres~ to: Grover C. Bagby, Jr., M.D., c/o Ann Irwin, Leukemia Research, V.A. Medical Center, Portland, OR 97207, U.S.A. *Supported by the Medical Research Service of the U.S. Veterans Administration, Portland, Oregon. "l'With Technical Assistance of Brenda Wilkinson. 571

572

GROVERC. BAGBY,JR TABLE 1. THE PRELEUKEMIC SYNDROME:DIAGNOSTICCRITERIA I. 2. 3. 4. 5. 6.

Anemia Megaloblastic or sideroblastic erythropoiesis. Either abnormal megakaryocytes or disorderly granulopoiesis Less than 5.% blasts in marrow Absence of Bt2 or folate deficiency No treatment with cytotoxic agents in the three months preceding diagnosis

h e r e i n , the r e s u l t s of s i m i l a r in v i t r o studies o n cells f r o m five p r e d n i s o l o n e - r e s p o n s i v e patients with the preleukemic syndrome.

MATERIALS

AND

METHODS

Patients and volunteers

The diagnosis of the prelcukemic syndrome, according to previously reported [4, 12.] criteria (Table 1), has been made in 74 patients since 1975. Marrow cells from all patients have been cultured rating semi-solid (CFU-C) [4,9, 13, 16, 17] and suspension [8] culture techniques. In eight (11%) of the 74 patients, cortisol or methylprednisolone enhanced granulocyte colony growth. The results of prednisone therapy in 5 of these patients have been reported [4]. Detailed in vitro studies on the mechanism of action of giucocorticmteroids in vitro have been carried out in a separate group of 5. Of the three patients not studied in detail, two had died and one was lost to follow-up. Patients 1, 2, and 3 in this paper represent patients 1, 4, and 5 respectively in our previously reported clinical series [4.]. During the period of the study, 31 healthy paid volunteers were studied concurrently for colony growth in agat. Signed informed consent was obtained in all patients and all volunteers. Preparation of marrow cells

Light density marrow cells were prepared and variably depleted of phagocytes (using a carbonyi iron-magnet technique), T-lymphocytes (using an E-rosette technique), and adherent cells (nylon wool columns) as previously described [2"]. When T-lymphocytes were depleted from a cell suspension, the T-depleted suspension was brought back to the original volume (the volume of the suspension prior to removal of T-cells) to avoid stem cell enrichment and to facilitate analysis of T-cell effects on granulopoiesis [2, 3]. Control cells were placed on Ficoli-Paque a second time. In three patients (patients 1, 2 and 3)' the T-cells separated from LDBMC were added back to an aliquot of autologous T-depleted LDBMC prior to plating. T-cells ranged from 10 to 50% of LDBMC. Phagocytes ranged from 12 to 43% of LDBMC. Marrow cell cultures

Both Afar and methyicellulose cultures were performed in 3 patients as previously described [4, 9, 13, 16, 17] using alpha medium (Flow Laboratories) with 15% fetal calf serum (Grand Island Biological Co.) in 0.9% methylcellulose and McCoy's 5a medium with 1570fetal calf serum (Grand Island Biological Co.) in 0.3% afar. In agar cultures, additives (steroids and LCM) were placed in a I ml 0.5% agnr underlayer (also containing complete medium and serum), whereas methylcellulose cultures were single I ml layers. LCM prepared according to a modification of the method of l~ove [10] served as our source of CSA. All cell L~tmpleswere cultured in the presence and absence of 10% LCM. All cultures were carried out in from three to five replicate 35 mm plastic culture dishes (Coming, NY). In each case, 1 ml aliquots of both LDBMC and T.~ell depleted LDBMC suspensions were placed in each plate. LDBMC were plated at concentrations of 2 x l0 s cells/ml, T-depleted plates contained fewer (by the number of T-cells in the LDBMC suspension) than 2 x l0 s cells. Thu& because T-cells ranged from 10 to 50% of LDBMC in this group of 5 patients the plated cell humbert ranged from 1 to 1.8 x l0 s. Colonies (>40 cells per clone) and clusters (>8 < 40 cells per clone) were counted after 14 days of culture in a humidified 7.5% CO, atmosphere. Colonies were singly picked out of agnr or methyicellulose and placed on glass slides for murphologic studies using the cytocentrifuge technique described by Dao et al. [5]. In individual patients, colony growth was considered to be different than controls if the P value was <0.05 according to the Mann-Whitney U test. SH-cortisol binding to the fetal calf serum in our system ranges from 11 to 38% [4]. Treatment of marrow cells prior to clonooenic culture

Cells from two patients were incubated in McCoy's 5a medium for I h with 10 -s M progesterone (Sigma, St. Louis, MO) or 10 -e M cortisol (hydrocortisone sodium succinate, Upjohn, Kalamazoo, MI) or both. The cells were washed thrice then placed in agar. Cells from 3 patients were incubated in complete medium for 1 h with ARA-C (Upjohn, Kalamazoo, MI) 50/~g/ml, or 10 -6 M cortisol, or both. The exposed cells were then washed thrice and plated. Colonies were counted after 14 days of culture under the conditions described above and

Glucocorticosteroidsand the preleukemicsyndrome

573

compared to colony growth of unexposedcells. The ARA-C data reflect CFU-C proliferative activity [1] and are expressed here as % CFU-C killed (Table 4) where: % CFU-C killed= 1009/o- ARA-Ccolony growth x 1 0 0 % . control colony growth To determine the effect of cortisol on CFU-C proliferation and survival in suspension culture, cells from patient 1 were placed in suspension culture (3 x 106 phagocyte depleted LDBMC/ml McCoy's 5a medium with 15% fetal calf serum) containing cortisol or LCM or both cortisol and LCM. Cells were serially harvested, washed, and plated in afar after 6, 12, 24 h and then dally through day 5. Recombination of autologous T-lymphocyteswith T-depletedLDBMC in a ratio of I lymphocyte:3 marrow cells prior to plating in aFar was performed using cells from patient 1, 2, and 3 as previously described [--2]. CSA assay

LDBMC (3 x 10e/mi)from patient 1 were cultured in suspension (M~._.oy's5a with 15~ fetal calf serum) for 14 days in the preeenoeand absence of 10-6 M cortisol. Conditioned medium was harvested on days 3, 5, and 14, and was stored unfiltered at -20°C. The conditioned media (0.05 and 0.1 ml/plate) were assayed for CSA activity against phatocyte depleted LDBMC (I x 10S/plate)from a normal volunteer. Colony growth of target cells did not occur in the absence of LCM. Colony growth was compared to that which was stimulated by conditioned medium from normal LDBMC harvested on the sixth day of culture. To avoid the potentially contaminating influenceof 10-7 M cortisol on target colony growth, progesterone (10-6 M) was added to each plate.

RESULTS Agar colony growth of light density bone marrow cells from normal volunteers ranges from 50 to 490 colonies and from 56 to 333 clusters/plate. T~lymphocyte depletion of L D B M C from normal volunteers has no effect on colony growth [2, 3]. We have previously reported consistent inhibition of granulocyte colony growth by cortisol (10 -6 M or greater) in normal volunteers and glucocorticosteroid unresponsive patients with the preleukemic syndrome [-2-4]. This inhibition is dose related and occurs equally in L D B M C and T-depleted L D B M C cultures. We have found that methylprednisolone and cortisol are equally inhibitory. Furthermore, in the three prednisolone-responsive patients so tested, both methylprednisolone and cortisol significantly enhanced colony growth and the dose response curves were congruent (data not shown). Both cortisol and methylprednisolone enhance colony growth when included in the agar medium for the duration of culture and in both patients tested (patients 1 and 2) both compounds enhanced colony growth after 1 h exposure of cells to steroid followed by washout and plating. Trypan blue exclusion viability was minimally altered (959/0) after 60 min exposure in cases 1 and 3. However, the cells were examined immediately after exposure and not after 6-12 h at which times T-cell viability should have been decreased. Enhancemerit of colony growth by cortisol of cells from patients 1 to 3 was documented in methylcellulose/alpha medium and agar with McCoy's 5a medium. Colony growth of L D B M C and T-depleted L D B M C in each of the 5 patients is shown in Table 2. Cortisol enhanced colony growth in all patients when tested with LDBMC. In patients 2-5, depletion of T-cells from L D B M C resulted in an enhancement of colony growth which equalled the maximum enhancement seen with cortisol when the steroid was included in culture with L D B M C . However, one T-lymphocytes were removed, cortisol failed to enhance colony growth further in patients 2-5. Because these observations suggested that cortisol sensitive T-cells were functioning to suppress granulopoiesis ['2, 3] in these patients, recombination experiments were performed wherein autologous T-lymphocytes were added back to T-depleted L D B M C from patients I-3 before and after exposure of purified T-cells to cortisol (10-~M) for 1 h. Purified T-cells were obtained by placing L D B M C that had been incubated with SRBC on a Ficoll-Paque cushion and obtaining a button of rosetted cells by centrifugation. The button was then treated with hypotonic lysis to yield T-cells. As shown in Table

574

GROVER C. BAOB¥, JR

O l.~rm

X

"O

I e~

n= O

E~ t~o ~

T

o

x~ ¢~I *"

k~

D

,¢

e~

"D

Glucocorticosteroids and the preleukemic syndrome

575

TASL~ 3. RECOMBINATIONOF T-LYMPHOCYTESWITH AUTOLOGOUSMARROW* TDLDBMC Recombined with eortisol exposed T-iymphocytes

Patient

LDBMC

ToDLDBMC

T-DLDBMC Recombined with T-lymphocytes

1

43 + 3

37 ± 4

48 + 3

65 5:6

2 3

0 95:2

15+5 42+3

0 9+2

13+4 70+10

*Experiments were performed in these three patients wherein autologous T-lymphocytes were added back T-lymphocytes depleted light density marrow cells before and after short term exposure (of T-cells) to cortisol (10 -s M). The T-lymphocyte: marrow cell ratio was: 1-3:4.

3, T-cells inhibited granulopoiesis in patients 2 and 3, but cortisol exposed T-lymphocytes did not. Autologous T-cells failed to inhibit granulopoiesis in patient 1. Serial agar cultures were performed in patients 1 and 2. Serial data in patient 1 have been published I'4]. A total of six agar cultures have been performed using cells from patient 1. At no time have we noted inhibitory T-cells during the course of his disease. In patient 2 (Fig. 1), in whom cortisol sensitive inhibitory T-cells have been found hematologic remission was noted only at doses (in vivo) of 160 rag/day. This patient became clinically resistant to even high doses of prednisone in August 1978. Subsequent studies (Fig. 1, March 1979) suggested and recombination experiments confirmed that the T-cells were cortisol resistant. We studied the effect of cortisol on CFU-C proliferation using the cytosine arabinoside technique 1'1]. As shown in Table 4, CFU-C killing was enhanced significantly (p < 0.05) by cortisol on 3 separate occasions using cells from patient number 1, but in patient 2, no such effect was seen. In our laboratory, mean CFU-C killing of LDBMC from normal e<0Ol

i^^

DATF"

T~mW )

Apr 78

,Am 78

Ju! 78

Mo¢ T9

mg

FIG. 1. Serial agar cultures of marrow cells from patient 2 in the presence (cross-hatched columns) and absence (white columns) of cortisol 10 -5 M. Dark shaded bars represent colony growth of T-cell depleted LDBMC. Bars and vertical lines represent means -1- S.D. colonies/2 × l0 s cells (except shaded bars which represent colonies/J2 × l0 s cells--T-cells]. Although nentropenia resolved in this patient with 60rag prednisone per day only 160rag per day achieved complete normalization of blood counts and baseline colony growth (June 1978). Even then, suppressor T-lymphocytes were detectable. In August, the patient's disease became refractory to prednisone and all subsequent in vitro studies have shown cortisol resistant suppressor T-cells.

576

GROVER C. BAGBY, JR TABLE 4. TmB EFFECT OF CORTISOL ON FRACTIONAL KILLING OF C F U - C eY A R A - C *

Patient l(a) (b) (c) (d) 2

Inhibitory T-cells Not Studied Absent Absent Absent Present

One hour incubation prior to culture % CFU-C Cortisol ARA-C killed Cortisol pins ARA-C

Medium (control) 23 55 42 18 64

+ + + + +

1 13 4 2 3

18 + 8 13 -1- 4 24 + 3 10 + 3 28 + 5

0 77 43 45 57 rJ, ! i," ,

36 58 66 42 108

+ + + + +

7 4 5 2 4

3 15 10 13 47

+ + + + +

% CFU-C killed

2 4 3 8 8

92 74 85 70 57

|

*Phagocyte depleted light density bone marrow cells from two patients were exposed to ARA-C (50 #8/mi), or cortisol (I0 -s M), or both for one hour prior to plating in afar. The reduction in colony growth (mean S.D.) with ARA-C treatment reflects the proliferative activity of CFU-C during the hour of exposure. In patient 1, cortisol enhanced CFU-C proliferation but in patient 2, it did not. b. Studied prior to prednisone therapy. c. Patient taking 60 m$ prednisone/day. d. Patient taking 20 mg prednisone/day.

volunteers using this technique ranges from 35 to 56% (n = 5). As shown in Fig. 2, cortisol enhanced CFU-C survival in suspension culture of cells from patient number one when phagocyte depleted LDBMC were cultured in the pre~ence of LCM. We hypothesized that in patient 1 cortisol acted on CFU-C directly or through a second population of cells which were induced by cortisol to enhance CFU-C proliferation. In an attempt to analyze the latter possibility, we studied the effect of cortisol on phagocyte depleted LDBMC and, as shown in Table 5, cortisol enhanced colony growth in the absence of CSA producing mononuclear phagocytes. This effect of cortisol was blocked by progesterone (Table 5). In addition, we measured CSA in medium conditioned by LDBMC from this patient and found that cortisol did not stimulate CSA production in vitro. These conditioned media also failed to enhance proliferation of CFU-C from autologous marrow cells (data not shown) in an ARA-C experiment which was performed in McCoy's 5a medium and 10 - 6 M progesterone. DISCUSSION In our continuing prospective study of patients with the preleukemic syndrome (bemopoietic dysplasia), we have found that 10~ of patients respond favorably to glucocorti-

i: !

Z

3

4

5

Fro. 2. Suspensions of phagocyte-depleted light density bone marrow cells (3 x 106/ml) from patient l were cultured without additives (B) or with cortisol 10 -6 M (A), LCM (C), and both cortisol and LCM (D). The samples of cells were washed and plated in agar over underlaycrs containing 10% LCM. Colonies were counted after 14 days in agar culture. Cortisol alone failed to enhance self replication (or survival) of CFU-C in suspension culture but significant enhancement occurred when both cortisol and LCM were present.

Glucocorticosteroids and the preleukemic syndrome

577

TXBLE 5. PROOF.Sl~ONE SLOCKS CORTISOLF.iq~I~CTIN PATIENTONE* First hour exposure Second hour exposure Progesterone (10 - s M) Cortisoi (10 - ° M) 0 + 0 +

0 0 + +

Coloniest

Clusterst

19+4 15 + 3 334-4 154-4

13+5 12 4- 3 254-4 114-3

"3 x 106 phagocyte depleted light density marrow cells were incubated for 60 rain with various combinations of cortisoi and progesterone, then the cells (2 x 10s per plate) were plated in agar. t M e a n + S.D.

costeroid therapy [4]. In addition, it has become apparent that in vitro techniques may aid in the identification of glucocorticosteroid responsive patients with the preleukemic syndrome as well as patients with a variety of other marrow failure states [2-4]. Furthermore, techniques wherein selected cell populations are depleted, treated with steroids or other pharmacologic agents, and recombined with target cells in clonogenic assays can elucidate both mechanisms of steroid effect and mechanisms of marrow failure in individual patients [2, 3]. We have employed clonogenic assays for granulocyte progenitor cells (CFU-C) in an attempt to define the mechanisms of action of giucocorticosteroids in those patients with the preleukemic syndrome who are responsive to prednisolone. In that population of patients, we have found that cortisol and methylprednisolone are equally effective in vitro, and that both steroids are capable of enhancing granulopoiesis in responsive patients when either methylcellulose or agar used as a matrix, and when RPMI 1640, McCoy's 5a medium, or alpha medium is used in the assay. We have confirmed our previously reported observation [4] that prednisolone alone does not stimulate colony growth in responsive patients, and we have also noted that prednisolone and cortisol (Table 5) responses are blocked by progesterone, a phenomenon described in other cell systems responsive to glucocorticosteroids [15]. The effects of glucocorticosteroids in patients 2-5 and patient 1 were clearly dissimilar. In patients 2-5, we have documented that cortisol sensitive T-cells in the marrow suppressed clonal granulopoiesis of autologous cells. For example, depletion of T-lymphocytes from the population enhanced granulopoiesis, and as shown in Table 2, cortisol and methylprednisolone enhanced granulopoiesis in these patients only when T-cells were present in the cultured population. In Table 3, recombination experiments confirmed (in patients 2 and 3) that the T-cells suppressed granulopoiesis and that they were cortisol sensitive. Our observations concerning the involvement of T-lymphocytes in suppressing granulopoiesis in these patients are probably not in vitro artifacts. Serial culture data acquired in studies on patient 2 are presented in Fig. 4 and document that during maximally effective prednisone therapy, the inhibitory activity of T-lymphocytes diminished. The observation in these four patients are similar to observations we have recently reported in other marrow failure states and documents, in this small subset of patients, the involvement of T-lymphocytes in the marrow failure of the preleukemic syndrome. We do not believe that T-lymphocytes account for all of the abnormalities seen in these patients. For example, although prednisone therapy in responsive patients accounts for a significant improvement in the effective hematopoiesis [.4] the morphologic abnormalities of dyshematopoiesis persist. Furthermore, overt, albeit oligoblastic, leukemia did develop in patient 3 in whom no suppressor T-cells were found during therapy. Thus, we persist in our tentative view of the preleukemic syndrome, as an intrinsic abnormality of hematopoietic cellular differentiation [12]. Nonetheless, the observations reported in

578

GROVERC. BAGBY,JR

these 4 patients document an additional involvement of the T-lymphocytes in a small subset of these patients. Whether the T-lymphocytes are part of the abnormal or normal hematopoietic clone is unknown. Detailed cytogenetic studies in prednisone responsive patients whose marrow cells exhibit a marker chromosome may be of value in this regard. None of our prednisone responsive patients exhibited marker chromosomes. Although we have suggested that the inhibitory effects of T-lymphocytes in marrow failure states (including patient 2, data not shown) is mediated in part by a soluble factor derived from the T-cells themselves [.2, 3] we have no data on the nature of the T-cell subset or the signals which may have effected initially the evolution of the inhibitory population. Although in this series we have seen no case of de-nero cortisol resistance of inhibitory T-cells, we have noted the acquisition during glucocorticosteroid therapy of inhibitory cell resistance not only in patient 2 of this series (Fig. 1) but in patients with other bone marrow failure states as well ['3]. In this setting, responses to non-steroidal immunosuppressive therapy may be favorable [.3]. Thus, the presence of inhibitory T-lymphocytes may account for the occasionally reported favorable response to such immunosuppressive therapy among patients with the preleukemic syndrome [,18]. Inhibitory T-lymphocytes were not found in patient 1 (Table 2 and 3), yet not only did cortisol and methylprednisolone enhance colony growth (Table 2 and 3), but the patient responded completely to prednisone therapy [4]. The effects of glucocorticosteroids in vitro in this patient were blocked by progesterone. These observations suggested that glucocorticosteroids stimulated directly the proliferation of CFU-C, suppressed a non-T inhibitor population, or enhanced the functional activity of a stimulator cell population. We tested some of these hypotheses. For example, as shown in Table 4, the % CFU-C killed during a 60 rain incubation with ARA-C doubled when cortisol was added to the pre-incubation mixture. Similar experiments in 3 normal volunteers (data not shown) and one prednisone-responsive preleukemic patient (Table 3) have shown no enhancement of CFU-C killing with cortisol. Figure 2 represents corroborative data suggesting that in the presence of LCM, CFU-C replication and survival were significantly enhanced by cortisol. Under various conditions, mononuclear phagocytes are capable of either enhancing (by providing CgA) or inhibiting (by producing prostaglandins E) clonal granulopoiesis [7, 11, 14]. Theoretically, enhancement of CFU-C proliferation by cortisol might therefore reflect suppression of prostaglandin E production or enhancement of CSA production. That cortisol enhanced colony growth of phagocyte-depleted LDBMC (see Table 5 and Fig. 2) suggests that an interaction of cortisol and the mononuclear phagocyte did not account for the effect of the steroid. Further, corroborative experiments on cells from this patient (Table 6) documented that cortisol did not enhance CSA production by LDBMC. These combined observations considered in the light of the rapidity of proliferative response of CFU-C to cortisol (2-3 fold enhancement within 60 rain) strongly suggest that in this patient cortisol enhanced granulopoiesis in vitro by stimulating directly the proliferation of CFU-C. Of additional interest, vis-il-vis in vitro/in rive correlations, is our finding (Table 4) that during maximally effective prednisone therapy (in rive) baseline colony growth increased from 18/2 × 105 cells to 75/2 × l0 s cells. At that dose, neither one hour (Table 4) nor continuous exposure [.4] to cortisol enhanced colony growth. Furthermore, ARA-C killed a larger fraction of CFU-C during maximally effective prednisone therapy than the fraction killed prior to therapy or during suboptimal therapy (Table 5). However, it should be recognized that while the in vitro and in rive observations in this patient are consistent with a direct stimulatory effect of steroids on CFU-C, we cannot be certain that an as yet unidentified intermediary cell population (non T-cell, non-phagocyte, non CFU-C) is involved in the CFU-C-steroid interaction.

Glucocorticosteroids and the preleukemic syndrome

579

TABLE 6. EFFECTOF CORTISOLON CSA PRODUt~"TIONBY L D B M C I~tOM PATIENT ONE*

Subject

Harvest day

1

Normal volunteer

Cortisol

Colonies/10 n Cells (mean 2: S.D.)

3

None

10 2:7

3 5

I0 -s M None

8 2:8 10 2:6

5 14 14 6 6

10 -e M None 10-~M None 10 -6 M

8 2:6 15 + 9 15 -I- 11 57 -t- 10 48 + 7

*3 × 106 LDBMC were cultured in suspension cultures for 3, 5, and 14 days. The conditioned media from these cultures were tested for CSA content by culturing in agar 10% conditioned medium and 2 × l0 s non-adherent light density marrow cells from a normal volunteer. Cortisol failed to enhance CSA production.

We have previously reported the potential clinical value of marrow cell culture techniques in identifying those patients with the preleukemic syndrome who will and those who will not respond favorably to glucocorticosteroid therapy !-4]. In this report, we have documented that both CFU-C and T-cell inhibitors of granulopoiesis may be the target cells of glueocorticosteroid action. The frequency of involvement of inhibitory T-cells of granulopoiesis in this steroid-responsive subset of preleukemic patients suggests that bone marrow failure is multifactorial and involves the immune apparatus or an intrinsic stem cell defect or both. Further detailed studies in responsive and unresponsive patients will be required to answer questions on the nature of stimuli which effect clonal expansion of the inhibitory T-cells, on the responsiveness to non-steroidal immunosuppressive therapy in the patients whose cultures show evidence of inhibitory T-cells, and on the overall effect of steroid therapy on the tendency of this syndrome to evolve to overt nonlymphocytie leukemia. It is clear that the preleukemic syndrome represents a pathophysiologieally heterogeneous group of hematopoietic disorders. In vitro studies which aid in the identification of responsive patients and mechanisms of marrow failure in this syndrome may aid in the design of rational arid individualized therapeutic approaches. Acknowledgement--The author thanks Ms. Pam Wilson for assistance in preparation of the manuscript.

REFERENCES I. BAGBYG. C. (1978) Stem cell (CFU-C) proliferation and emergence in a case of chronic granulocytic leukaemia: the role of the spleen. Stand. J. Haemat. 20, 13. 2. BAGBYG. C. & GABOURELJ. D. (1979) Neutropenia in three patients with rheumatic disorders. Suppression of granulopoiesis by cortisol-sensitive thymus-dependent lymphocytes. J. din. Invest 64, 72. 3. BAOSYG. C., GOODmOHTS. H., MOONEYW. M., et al. Prednisone responsive aplastie anemia: a mechanism of glucocorticoid action. Blood 54, 322. 4. BAGBYO. C., GA~mR~. J. D. & LINM^NJ. W. (1980) Glucocorticoid therapy in the preleukemic syndrome (hemopoietic dysplasia). Identification of responsive patients using in vitro techniques. Ann. intern. Med. 92, 55. 5. DAO C. D , ML~rCxt.FR., ~ t m R., et al. (1977) Normal human bone marrow cultures in vitro: cellular composition and maturation of the gsannlocytic colonies. Br. J. Haemat. 3'7, 127. 6. ELIASL. E. & G R ~ S m O P. (1977) Divergent patterns of marrow cell suspension culture growth in the myeloid leukemias: correlation of in vitro findings with clinical features. Blood .~0, 263. 7. GOL~ D. W. & C u ~ M. J. (1972) Identification of colony stimulating cells in human peripheral blood. J. Clin. Invest. 51, 2981. 8. GOLDED. W. & CIJNe M. D. (1973) Human preleukemia: identification of a maturation defect in vitro. N. Engl. J. Med. 288, 1083.

580

GRGV~ C. B^cBY, JR

9. GX~NS,~O P. L., NICHOLSW. G. & SCHRIERS. L. (1971) Granulopoiesis in acute myeloid leukemia and preleukemia. N. Engl. J. Med. 284, 1225. 10. Iscov~ N. N., SL~NNJ. S., TILl. J. E. et aL (1971) Colony formation by normal and leukemic human marrow cells in culture. Effect of conditioned medium from human leucocytes. Blood 37, l. I I. KURLANDJ. & MOOI~ M. A. S. (1977) Modulation of hemopoiesis by prostaglandins. Exp. Hemat. 5, 375. 12. LINMANJ. W. & BAGBYG. C. (1978) The preleukemic syndrome (hemopoietic dysplasia). Cancer 42, 854. 13. MILNER(3. R., TESTAN. (3., G u Y C. G., et al. (1977) Bone marrow culture studies in refractory cytopenia and smouldering leukemia. Br. J. Haemat. 3~ 251. 14. MOORI~M. A. S. & WILLIAMSN. (1972) Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic tissue. J. Cell. Physiol. 80, 195. 15. Rouss~u G. G., B.~crl~ J. D. & TOI~KINSG. M. (1972) Glucocorticoid receptors: relations between steroid binding and biological effects. J. molec. Biol. 67, 99. 16. SENNJ. S. & PIN--TON P. H. (1972) Defective in vltro colony formation by human bone marrow preceding overt leukemia. Br. J. Haemat. 23, 277. 17. VERMAD. S., SPITZERG., DICI~ K. A., et al. (1979) In vitro agar culture patterns in preleukemia and their clinical significance. Leukemia Res. 3, 41. 18. Z~VAS C. G., GI~RY C. (3. & OL~_~-~KYS. (1974) Sideroblastic anemia treated with immunosuppressive therapy. Blood 44, 117.