59
EFFECT OF SODIUM SALICYLATE ON HUMAN AND MOUSE GRANULOPOIESIS IN VITRO J. D. GABOUREL, M . A. S. MOORE, G. C. BAGBY, Jr., and G . H. DAVIES Sodium salicylate inhibited generation of granulocyte and macrophage colonies when added to soft agar cultures of mouse or human bone marrow cells (CFUc) containing colony-stimulating factor (CSF). This effect was dose-dependent with over 90%inhibition at 48 mg%. The salicylate effect was not decreased with increasing concentrations of CSF, but inhibition was reversed when salicylate-treated CFUc were washed with drug-free medium before plating. CSF production was not inhibited by salicylate.
Recently a number of drugs, phenobarbital ( I ) , diphenylhydantoin (2), halothane and other anesthetics (3-5), salicylates (6-8), and oral contraceptives (9) have From the Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Melbourne, Victoria, Australia, and the Department of Pharmacology. University of Oregon Health Sciences Center, Portland, Oregon. Supported by USPHS NIH Grant C A 14360 and by a grant from the Alma Johnson Foundation Trust. J. D. Gabourel, Ph.D.: Visiting Scientist, Walter and Eliza Hall Institute of Medical Research, Professor of Pharmacology, University of Oregon, and recipient of USPHS Grant CA/GM 14360. M. A. S. Moore, D.Phil.: Head, Laboratory of Developmental Biology, Cancer Research Unit, Walter and Eliza Hall Institute of Medical Research (currently a member of the Sloan-Kettering Institute for Cancer Research, and Head, Gar Reichman Laboratory for Advanced Cancer Research); G . C. Bagby, Jr., M.D.: Instructor, Division of Hematology and Medical Oncology, University of Oregon; G . H. Davies, A.I.S.T. Diploma: Research Associate in Pharmacology, University of Oregon. Address reprint requests to J. D. Gabourel, Ph.D., Department of Pharmacology, University of Oregon Health Sciences Center, Portland, Oregon 97201. Submitted for publication February 10, 1976; accepted July 2, 1976.
Arthritis and Rheumatism, Vol. 20, No. I (January-February 1977)
been shown to suppress the mitogenic response of circulating lymphocytes to phytohemagglutinin (PHA). Salicylates have also been reported to inhibit the mixed lymphocyte response ( M L R ) between allogeneic human lymphocytes ( 6 ) . On the basis of such experiments it has been suggested that these drugs may suppress the cellular arm of the immune system. Optimal immune responses also require the participation of macrophages (mononuclear) and polymorphonuclear leukocytes (10). The authors have investigated the effects of one of these drugs, sodium salicylate, on leukocyte proliferation by testing its effects on the generation of granulocyte and macrophage colonies from murine and human bone marrow cells in soft agar cultures containing colony- stimulating factor (CSF). Each colony was presumed to have originated from a single bone marrow cell, and the parent cells have been designated colony-forming units in culture (CFUc) (1 l). Drug effects on proliferation of multipotential bone marrow stem cells in vivo were also investigated, by means of the spleen colony assay developed by Till and McCulloch (12). Again each colony was thought to arise from a single stem cell, and these multipotential stem cells have been designated colony forming units in spleen (CFUs).
MATERIALS AND METHODS Animal Studies Animals. F e m a l e (C57 B1/6J X C B A / H ) F, WEHl mice, aged 65-100 d a y s a n d weighing 20-25 g , were used
60
GABOUREL ET AL
Table 1. Effect of Sodium Salicylate on CSF Stimulation of Mouse Bone Marrow Cells in Vitro Salicylate Concentration (mg%)
O* 48 32 16
8 4
Colonies/105 Cellst Experiment 2 Experiment I
113 2 21 57 93
f 13 f 2$ f 5$ f 14$ f 13 120 f 9
78 f 5 8 f 5$ 27 f 2$ 5 1 f 13$ 7 0 f 10 -
Sodium salicylate (4-48 mg% final concentration) was added to liquid agar cultures of mouse bone marrow cells. Cultures were incubated for 7 days at 37°C. and the number of colonies was enumerated by visual observation with a dissecting microscope. * Control. t Mean of quadruplicate assays f SD. P < 0.01 compared with control; student's two-tailed t test.
throughout except for one experiment to assess C S F production, which used female BALB/c mice (Simonsen Labs) aged 74-75 days and weighing 16-20 g. Procedures. Mouse granulocyte and macrophage colonies were generated in modified Eagles medium containing 0.3% agar and mouse serum CSF as previously described by Metcalf and Moore (1 1 ). Each culture contained 0.05 ml of a 1 :6 dilution of serum taken from mice 3 hours after injection with 5 pg E coli endotoxin (Difco) as a source of CSF. A just suboptimal dose of C S F was incorporated directly into the liquid agar medium. Bone marrow cells were added so that each milliliter contained 50,000 cells. Replicate 1 -ml aliquots of the resulting cell suspension were pipetted into 35-mm petri dishes. The appropriate concentration of drug in 0.1 ml was added and mixed thoroughly before gelling occurred. Addition of drug did not alter the pH of the medium. The cultures were then incubated for 7 days a t 37"C, after which each culture was examined and the number of colonies determined. All assays were done in quadruplicate. Mouse Spleen Colony Assay in Vivo. The method used was essentially that developed by Till and McCulloch (12). In brief, 106 syngeneic bone marrow cells were injected intravenously into lethally irradiated mice. The total dose of 900 R was given in two doses of 450 R each, 24 hours apart (13). Seven days later the recipient spleens were removed and fixed in Bouin's solution, and the number of macroscopically visible nodules or colonies on the surface of the spleen was determined. Each nodule is considered to be the product of proliferation of a single multipotential stem cell (CFUs).
Human Studies Colony stimulating activity (CSA) was prepared as peripheral blood leukocyte-conditioned medium according to the method of Iscove et al (14), and 0.05-0.20 ml CSA was added to 35-mm plastic culture dishes (Falcon). One milliliter of McCoy's 5A medium (GIBCO) containing 0.5% agar was added to each dish, allowed to gel, and incubated for 20 hours before use as an underlayer.
The method of Golde and Cline (15) was used to prepare 2 X 10' cells/ml suspended in 0.3% agar (in McCoy's 5A medium) from bone marrow aspirates from 4 hospitalized patients and 1 normal volunteer. One milliliter of this suspension was placed over each underlayer and incubated at 37.5"C in a 7.5% CO, and 95% humidity atmosphere for 10 days, at which time colonies (>50 cells per aggregate) were counted.
RESULTS Sodium salicylate inhibited generation of granulocyte and macrophage colonies when added to agar cultures of mouse bone marrow cells containing CSF (Table 1 ). Significant inhibition occurred with that concentration range found in plasma during the therapy of rheumatoid arthritis ( 1 6). Salicylate inhibition of colony formation was demonstrable only when the drug was present in the agar culture medium and was not seen when bone marrow cells from salicylate-treated mice were cultured in the absence of drug (Table 2). Furthermore, no difference in total femoral shaft counts or morphology (bone marrow smears) could be detected after such 5-day treatment in vivo. Sodium salicylate was also tested for its effects on the formation in vivo of spleen colonies (nodules) when low numbers of syngeneic bone marrow cells were injected into lethally irradiated mice. The numbers of CFUs found in control and salicylate-treated mice are shown in Table 3. No significant inhibition was seen. Mouse CSF production in response to intravenous injection of 5 pg E coli endotoxin was assayed in control and salicylate-treated mice (Table 4). Elevation of serum CSF in control endotoxin-treated mice was less marked than previously reported (1 1); however Table 2. Effect
01Salicylale Pretreatment in Viuo on CSF Stimulation of Mouse Bone Marrow Cells in Viuo
Treatment* Control Salicylate
Colonies/lP Cellst
Cells/Shaft x lo-ei
I84 f 48 175 f 47
19.0 f 1.8 22.9 f 3.3
"Bone marrow cells were taken from mice that had been injected intraperitoneally with 5 mg sodium salicylate in 0.5 ml of 0.5% NaCl four times daily for 5 days. These cells were cultured for 7 days in soft agar media containing C S F in the absence of salicylate, and the number of colonies was enumerated as described previously. Blood levels in mice reached 37-38 mg% I hour after each of three sequential injections spaced 4 hours apart. That drug accumulation occurred suggested that sodium salicylate is rapidly eliminated. t Results are the means from 6 mice f SD.
61
SALICYLATE AND LEUKOCYTE PROLIFERATION
Table 3. Salicylate Effect on Spleen CFUs CFUs/Spleen f SD (m) Treatment* Control Salicylate
EXPI t
EXP2t
28 f 5 (8) 1 8 f 8 (10)
17f4(11) 1 5 f 3 (11)
EXP 3$ 20f6(9) 18 f 3 (9)
* Syngeneic bone marrow cells ( I @ ) were adoptively transferred into irradiated mice. Spleens were excised and assayed for CFUs on day 7. No colonies were seen on spleens of mice that did not receive injections of bone marrow cells. t (C57BI X CBA) F, mice were irradiated with 900 R in two 450 R doses 24 hours apart. Salicylate-treatment mice received 30 mg sodium salicylate per day in four equal intraperitoneal doses (Exp I ) or in three equal doses (Exp 2). Drug treatment started 2 hours before adoptive transfer and was continued for 4 days. $. Recipient BALB/c mice were irradiated with a single 510 R dose. Both donor and recipient mice were pretreated with sodium salicylate for 7 days prior to adoptive transfer. Treatment of recipient mice was continued for an additional 7 days until sacrifice. Each mouse received 20 mg sodium salicylate per day in 4 equal intraperitoneal injections. salicylate treatment did not suppress CSF production and indeed appeared to enhance significantly the serum levels. Salicylate also suppressed human granulocyte and macrophage colony formation induced by CSA in vitro (Table 5 ) . This response was somewhat more variable than that seen with inbred mice (Table 1). Overall, however, the inhibition appeared to be dose-dependent for any individual patient. N o inhibition occurred when bone marrow cells were preincubated with salicylate in vitro for 2-5 hours and then washed and plated over an underlayer containing CSA (Table 6). None of the differences between control groups and comparable groups preincubated with salicylate for either 2 or 5 hours was significant at P < 0.2 (student's two-tailed r test).
DISCUSSION It has been shown that salicylates inhibit some lymphocyte functions and further suggested that they may possess immunosuppressant activity (6,17). The results presented here show that concentrations of salicylate similar to those inhibiting lymphocyte transformation (8-48 mg%) also inhibit CSF-induced proliferation of mouse CFUc in vitro when the drug is present continuously in the culture medium (Table 1). CSF used to stimulate mouse cultures is a glycoprotein with .a molecular weight of approximately 20,000. It is highly species-specific with effective dose levels in culture of the M (11). There is considerable order of lo-" to indirect evidence that CSF may also act in vivo as a
regulator of granulopoiesis and monocyte production. Other factors are involved, however, and the exact role of CSF in granulopoiesis in vivo remains to be established. Salicylate also inhibits human CFUc response to CSA in vitro with a similar dose response relationship (Table 4). CSA from cultured human leukocytes is less well characterized than mouse CSF and is stimulatory to both mouse and human CFUc. Although it has been established that salicylates inhibit the proliferative response of CFUc to CSF (or CSA) in vitro, it should be emphasized that the precise nature and location of the salicylate effect remains to be identified. Salicylate concentrations, used to inhibit generation of granulocyte and macrophage colonies in vitro, are similar to those found in salicylate-treated rheumatoid arthritis patients, but direct comparison to in vivo plasma concentrations is clouded because of possible differences in drug protein binding. Salicylate is not appreciably bound to plasma proteins in the mouse; 7% bound at 5 mg% and 5% bound as total plasma level reached 15 mg% (17-19). This result is in contrast to that in man, where protein binding is 75% at salicylate concentrations of 5 mg%, and decreases to 50% at 50 mg%. Both mouse and human bone marrow culture media contain 15% fetal calf serum. Salicylate inhibition of colony formation was demonstrable only when the drug was present in the soft Table 4. Salicylate Effect on E coli Endotoxin-Induced Mouse CSF Production Treatment*
CSF Units/O. I of Serum f SDt
Control Salicylate
8 f 3 68 f 14
* Twenty-three BALB/c mice were split into 2 groups; 12 mice received 0.5% NaCl and 11 mice received 250 mg sodium salicylate per kg per injection. Mice were injected three times daily for 30 days. Drinking water was withheld during the day and mice were given distilled water or sodium salicylate (3 mg/ml) in distilled water to drink overnight. Salicylate-treated mice consumed an extra 6.57 f 1.32 (SD) mg/ mouse/day via this route. The volume of fluid consumed was not significantly different for the two groups. t Mice were bled 3 hours after intravenous injection of 5 pg E coli endotoxin (DIFCO). Pooled blood samples were allowed to clot, and serum was removed and dialyzed against 0.02% sodium azide in distilled water for 24 hours and then against two changes of distilled water for an additional 48 hours. The serum was removed from the dialysis sacks, sterilized by millipore filtration, and assayed for colony stimulating activity with 7.5 X 10' C57B1/6 bone marrow cells. Serum was titrated in quadruplicate cultures over the range 1 : 1 to I :64 by the standard technique of Metcalf and Moore ( 1 I ) . CSF activity is expressed as the number of colonies stimulated in the linear portion of the dose response curve: 1 unit of CSF = 1 colony stimulated.
GABOUREL ET AL
62
Table 5. Effecf of Salicylate on Human Bone Marrow CSA Induced Colony Formation in Vitro
No. of Colonies/2 Patient
Diagnosis
1
Juvenile chronic myelogenous leukemia Agnogenic myeloid metaplasiaS Hodgkin's lymphoma Normal volunteer Neutropeniag Preleukemia
2 3 4 5 6
Nucleated Marrow Cells f SD* Salicylate 32 mg%t 48 mg%t
X l@
Control f SD
16 mg%t
251 rf: 123
268 f 34
101 f 15
4 0 f 15
2 f 2 35 f 10
251 17f8 6410 201 f 90 5 f 2
2.5 f 3 1715 2 f 1.4 171 f 2 7 2*0
4 9 f 10 3 0 f 10 49 f 6 280 f 9 1 2 f 10
-
270 f 41 13 f 7
*A
standard amount of CSA (100 pl) was added to each plate. Data are averages of colony counts from three to six plates. t Final salicylate concentration in the overlayer. $ C F U c obtained from spleen at time of splenectomy. 5 Marrow obtained during recovery phase.
agar culture and was not seen when bone marrow cells from salicylate-treated mice were cultured in the absence of drug (Table 2). Furthermore, no difference in total femoral shaft counts or morphology (bone marrow smears) could be detected. The fact that pretreatment of mice with large doses of sodium salicylate did not suppress colony formation in vitro might be explained by the reversibility of the salicylate-induced inhibition. Salicylate effects on lymphocytes in vitro have been shown to be reversible when the drug is removed by washing ( 6 ) .The present authors have also demonstrated that the effects on human bone marrow cells in vitro are similarly reversible (Table 5 ) . Thus washing of bone marrow cells in preparation for liquid agar culture may have removed or diluted any drug present as a result of in vivo treatment. The ability to reverse the salicylate effect by washing indicates that salicylate is not cytotoxic to human bone marrow cells and that the effects on granulopoieses are reversible. Chronic therapy with high doses of sodium salicylate for 30 days did not inhibit CSF production in response to intravenous injection of E cofi endotoxin, and indeed it appeared to enhance the response (Table 4). In this context Kurland and Moore (20,21) have observed that activated mouse peritoneal macrophages are a potent source of both CSF and prostaglandins of the E series. The latter are very potent inhibitors of CFUc in vitro, and physiologic concentrations of PGE, may serve to limit macrophage production of CSF in response to such activating agents as endotoxin. Because indomethacin treatment inhibits prostaglandin synthesis and enhances in vivo and in vitro CSF production (21 ), it is possible that the increased CSF production in sa-
licylate-treated mice is due to a similar mechanism. Kurland and Moore ( 2 2 ) have also shown that E-type prostaglandins directly inhibit the proliferation of committed granulocyte macrophage progenitor cells in vitro and that the inhibition can be overridden by increased concentrations of CSF. Because E-type prostaglandins have been demonstrated to suppress directly CFUc proliferation and to interfere with CSF production, it is unlikely that salicylate inhibition of prostaglandin synthetase could account for the suppressive salicylate effects described here. It is also interesting that acetylsalicylic acid (aspirin) and sodium salicylate are equally effective against experimental inflammation and in the treatment of rheumatoid arthritis (23), even though sodium salicylate has a much weaker inhibitory effect on prostaglandin synthesis in vitro (24). Furthermore, Smith et af (25) have shown that aspirin but not sodium salicylate caused a Table 6. Reversibility of Salicylate Inhibition of Colony Formation in Vitro afier Cell Washing Treatment Control (2 h r )
Amount of CSA Added 50 100
Salicylate (2 hr)
Salicylate (5 hr)
200 50 I00 200 50 I00 200
Colonies/2 X lo1 nucleated BM cells* 42 f 21 52 f 27 6 4 f 15 58 rf: 6 58 f 6 I21 f 55 76 f 31 40 f 6 81 f: 25
* Data are averages of colony counts for three plates
rf:
SD.
SALICYLATE AND LEUKOCYTE PROLIFERATION
significant reduction in the potentiation of carrageenininduced paw edema when arachidonic acid was given concurrently. Therefore the antiinflammatory effects of sodium salicylate may be mediated by a mechanism other than prostaglandin synthetase inhibiton. Because aspirin is rapidly converted to salicylic acid in vivo (26), it may be that it exerts part of its antiinflammatory activity by a second mechanism that is mediated by salicylate anion and does not involve inhibition of prostaglandin synthesis. Salicylate effects on the ability of certain adoptively transferred primitive hemopoietic stem cells to form macroscopically visible colonies of differentiating erythroid, granulocytic, or megakaryocytic cells in spleens of lethally irradiated mice were also investigated. N o significant effect on the number of CFUs per spleen could be detected when mice were pretreated with sodium salicylate prior to adoptive transfer and treatment continued during the period of in vivo culture (Table 3). It should be recognized, however, that in vivo CFUs are pluripotent stem cells, whereas the in vitro colony assay detects committed granulocyte monocyte progenitor cells (1 1 ). The lack of detectable effects on CFUs proliferation in the intact mouse may reflect the authors’ inability to maintain relatively constant high blood levels. No salicylate accumulation was found in mouse blood after three sequential intraperitoneal injections of 5 mg of sodium salicylate spaced 4 hours apart (Table 2 legend), a result suggesting almost complete elimination of an injected dose in the 4-hour period. In contrast, salicylate accumulation occurs in man during continuous therapy. When aspirin is given every 8 hours, plateau drug concerltrations are reached after 6 to 9 doses of 500 mg each or after 20 doses of 1000 mg each (27,28). Thus it is likely that high blood levels are better maintained in humans than in mice. It is unclear, however, whether chronic high-dose salicylate therapy results in suppression of granulopoiesis in man. In summary, salicylate is suppressive to CSF- to CSA-induced granulocyte and macrophage proliferation in agar. It is unlikely that these effect? are mediated through inhibition of prostaglandin synthetase. As yet the authors have been unable to demonstrate such suppression in vivo, and further studies in vivo are necessary to substantiate the possible clinical relevance of the in vitro effects described here.
ACKNOWLEDGMENT The authors are grateful to Ms. J. Yates for expert technical assistance.
63
REFERENCES I . Park SK, Brody JT: Suppression of immunity by phenobarbital. Nature [New Biol] 233:181-182, 1971 2. Sorrel1 TC, Forbes IJ, Burness F R , et al: Depression of immunological function in patients treated with phenytoin sodium (sodium diphenylhydantoin). Lancet 1 : 12331235, 1971 3. Bruce DL: Halothane inhibition of phytohemagglutinin transformation of lymphocytes. Anesthesiology 36:201205, 1972 4. Viljunen M K , Kanto J, Vapaavurori M: Immunosuppression by halothane. Br Med J 3:499-500, 1973 5. Wingard DW, Lang R, Humphrey LJ: Effect of anesthesia on immunity. J Surg Res 7:430-432, 1967 6. Opelz G, Terasaki PI, Herata AA: Suppression of lymphocyte transformation by aspirin. Lancet 2:478-480, I973 7. Loveday C, Eisen V: Salicylates and suppression of lymphocyte transformation. Lancet 2:676, 1973 8. Schneider W, Pappas A, Scheurlen PG: Salicylates and suppression of lymphocyte transformation. Lancet 2:676677, 1973 9. Barnes EW, Loudon NB, MacCuish AC, et al: Phytohemagglutinin-induced lymphocyte transformation and circulating auto-antibodies in women taking oral contraceptives. Lancet 1398-900, 1974 10. Nelson DS: Immunity to infection, allograft immunity and tumor immunity: parallels and contrasts. Transplant Rev 19:226-254, 1974 11. Metcalf D, Moore MAS: Haemopoietic cells. Amsterdam and London, North Holland Publishing, New York, American Elsevier, 1971, pp 37-40, 383-419 12. Till JE, McCulloch EA: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Res 14:2 13-222, 1961 13. Golub ES: Brain-associated stem cell antigen: an antigen shared by brain and hemopoietic stem cells. J Exp Med 136:369-374, 1972 14. Iscove WW, Senn JS, Till JE, et al: Colony formation by normal and leukemic human bone marrow cells in culture: effect of conditioned medium from human leukocytes. Blood 37:l-5, 1971 15. Golde DW, Cline MJ: Growth of human bone marrow in liquid culture. Blood 41:45-47, 1973 16. Woodbury DM: Analgesic-antipyretics, anti-inflammatory agents and inhibitors of uric acid synthesis, The Pharmacological Basis of Therapeutics. Fourth Edition. Edited by LS Goodman, A Gilman. New York, Macmillan, 1970, p 328 17. Forbes IJ, Smith JL: Effects of anti-inflammatory drugs on lymphocytes. Lancet 2:334-337, 1967 18. Davison C: Salicylate metabolism in man. Ann NY Acad Sci 179:249-268, 197 1 19. Sturman JA, Smith MGH: The binding of salicylate to
GABOUREL ET AL
20.
21. 22. 23. 24.
plasma proteins in different species. J Pharmacol I9:621-623, 1967 Moore MAS, Kurland J: Regulation of granulopoiesis, Proceedings of the Second International Conference on Cell Differentiation. Edited by D Viza. ASP Biological and Medical Press, 1976 Kurland J, and Moore MAS: Manuscript in preparation Kurland J, Moore MAS: Effect of prostaglandin on hemopoietic progenitor cells. Fed Proc 34:251, 1976 Collier HOJ: A pharmacological analysis of aspirin. Adv Pharmacol Chemother 7:333-405, 1969 Vane JR: Inhibition of prostaglandin synthetase as a mechanism af action for aspirin-like drugs. Nature [New Biol] 231:232-235, 1971
25. Smith MJH, Ford-Hutchinson AW, Elliott PNC: Prostaglandiqs and the anti-inflammatory activities of aspirin and sodium salicylate. J Pharm Pharmacol 27:473-478, 1975 26. Levy G , Leonards JR: Absorption, metabolism and excretion of salicylates in MJH Smith and PK Smith, The Salicylates. New York, Wiley, 1966, pp 5-48 27. Paulus HE, Siege1 M, Morgan E: Variation of serum concentrations and half-life of salicylate in patients with rheumatoid arthritis. Arthritis Rheum 14:527-532, 1971 28. Levy G , Tsuchiya T: Salicylate accumulation in man. N Engl J Med 287:430-432, 1972
EFFECT OF SODIUM SALICYLATE ON HUMAN AND MOUSE GRANULOPOIESIS IN VITRO J. D. GABOUREL, M . A. S. MOORE, G. C. BAGBY, Jr., and G . H. DAVIES Sodium salicylate inhibited generation of granulocyte and macrophage colonies when added to soft agar cultures of mouse or human bone marrow cells (CFUc) containing colony-stimulating factor (CSF). This effect was dose-dependent with over 90%inhibition at 48 mg%. The salicylate effect was not decreased with increasing concentrations of CSF, but inhibition was reversed when salicylate-treated CFUc were washed with drug-free medium before plating. CSF production was not inhibited by salicylate.
Recently a number of drugs, phenobarbital ( I ) , diphenylhydantoin (2), halothane and other anesthetics (3-5), salicylates (6-8), and oral contraceptives (9) have From the Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Melbourne, Victoria, Australia, and the Department of Pharmacology. University of Oregon Health Sciences Center, Portland, Oregon. Supported by USPHS NIH Grant C A 14360 and by a grant from the Alma Johnson Foundation Trust. J. D. Gabourel, Ph.D.: Visiting Scientist, Walter and Eliza Hall Institute of Medical Research, Professor of Pharmacology, University of Oregon, and recipient of USPHS Grant CA/GM 14360. M. A. S. Moore, D.Phil.: Head, Laboratory of Developmental Biology, Cancer Research Unit, Walter and Eliza Hall Institute of Medical Research (currently a member of the Sloan-Kettering Institute for Cancer Research, and Head, Gar Reichman Laboratory for Advanced Cancer Research); G . C. Bagby, Jr., M.D.: Instructor, Division of Hematology and Medical Oncology, University of Oregon; G . H. Davies, A.I.S.T. Diploma: Research Associate in Pharmacology, University of Oregon. Address reprint requests to J. D. Gabourel, Ph.D., Department of Pharmacology, University of Oregon Health Sciences Center, Portland, Oregon 97201. Submitted for publication February 10, 1976; accepted July 2, 1976.
Arthritis and Rheumatism, Vol. 20, No. I (January-February 1977)
been shown to suppress the mitogenic response of circulating lymphocytes to phytohemagglutinin (PHA). Salicylates have also been reported to inhibit the mixed lymphocyte response ( M L R ) between allogeneic human lymphocytes ( 6 ) . On the basis of such experiments it has been suggested that these drugs may suppress the cellular arm of the immune system. Optimal immune responses also require the participation of macrophages (mononuclear) and polymorphonuclear leukocytes (10). The authors have investigated the effects of one of these drugs, sodium salicylate, on leukocyte proliferation by testing its effects on the generation of granulocyte and macrophage colonies from murine and human bone marrow cells in soft agar cultures containing colony- stimulating factor (CSF). Each colony was presumed to have originated from a single bone marrow cell, and the parent cells have been designated colony-forming units in culture (CFUc) (1 l). Drug effects on proliferation of multipotential bone marrow stem cells in vivo were also investigated, by means of the spleen colony assay developed by Till and McCulloch (12). Again each colony was thought to arise from a single stem cell, and these multipotential stem cells have been designated colony forming units in spleen (CFUs).
MATERIALS AND METHODS Animal Studies Animals. F e m a l e (C57 B1/6J X C B A / H ) F, WEHl mice, aged 65-100 d a y s a n d weighing 20-25 g , were used
60
GABOUREL ET AL
Table 1. Effect of Sodium Salicylate on CSF Stimulation of Mouse Bone Marrow Cells in Vitro Salicylate Concentration (mg%)
O* 48 32 16
8 4
Colonies/105 Cellst Experiment 2 Experiment I
113 2 21 57 93
f 13 f 2$ f 5$ f 14$ f 13 120 f 9
78 f 5 8 f 5$ 27 f 2$ 5 1 f 13$ 7 0 f 10 -
Sodium salicylate (4-48 mg% final concentration) was added to liquid agar cultures of mouse bone marrow cells. Cultures were incubated for 7 days at 37°C. and the number of colonies was enumerated by visual observation with a dissecting microscope. * Control. t Mean of quadruplicate assays f SD. P < 0.01 compared with control; student's two-tailed t test.
throughout except for one experiment to assess C S F production, which used female BALB/c mice (Simonsen Labs) aged 74-75 days and weighing 16-20 g. Procedures. Mouse granulocyte and macrophage colonies were generated in modified Eagles medium containing 0.3% agar and mouse serum CSF as previously described by Metcalf and Moore (1 1 ). Each culture contained 0.05 ml of a 1 :6 dilution of serum taken from mice 3 hours after injection with 5 pg E coli endotoxin (Difco) as a source of CSF. A just suboptimal dose of C S F was incorporated directly into the liquid agar medium. Bone marrow cells were added so that each milliliter contained 50,000 cells. Replicate 1 -ml aliquots of the resulting cell suspension were pipetted into 35-mm petri dishes. The appropriate concentration of drug in 0.1 ml was added and mixed thoroughly before gelling occurred. Addition of drug did not alter the pH of the medium. The cultures were then incubated for 7 days a t 37"C, after which each culture was examined and the number of colonies determined. All assays were done in quadruplicate. Mouse Spleen Colony Assay in Vivo. The method used was essentially that developed by Till and McCulloch (12). In brief, 106 syngeneic bone marrow cells were injected intravenously into lethally irradiated mice. The total dose of 900 R was given in two doses of 450 R each, 24 hours apart (13). Seven days later the recipient spleens were removed and fixed in Bouin's solution, and the number of macroscopically visible nodules or colonies on the surface of the spleen was determined. Each nodule is considered to be the product of proliferation of a single multipotential stem cell (CFUs).
Human Studies Colony stimulating activity (CSA) was prepared as peripheral blood leukocyte-conditioned medium according to the method of Iscove et al (14), and 0.05-0.20 ml CSA was added to 35-mm plastic culture dishes (Falcon). One milliliter of McCoy's 5A medium (GIBCO) containing 0.5% agar was added to each dish, allowed to gel, and incubated for 20 hours before use as an underlayer.
The method of Golde and Cline (15) was used to prepare 2 X 10' cells/ml suspended in 0.3% agar (in McCoy's 5A medium) from bone marrow aspirates from 4 hospitalized patients and 1 normal volunteer. One milliliter of this suspension was placed over each underlayer and incubated at 37.5"C in a 7.5% CO, and 95% humidity atmosphere for 10 days, at which time colonies (>50 cells per aggregate) were counted.
RESULTS Sodium salicylate inhibited generation of granulocyte and macrophage colonies when added to agar cultures of mouse bone marrow cells containing CSF (Table 1 ). Significant inhibition occurred with that concentration range found in plasma during the therapy of rheumatoid arthritis ( 1 6). Salicylate inhibition of colony formation was demonstrable only when the drug was present in the agar culture medium and was not seen when bone marrow cells from salicylate-treated mice were cultured in the absence of drug (Table 2). Furthermore, no difference in total femoral shaft counts or morphology (bone marrow smears) could be detected after such 5-day treatment in vivo. Sodium salicylate was also tested for its effects on the formation in vivo of spleen colonies (nodules) when low numbers of syngeneic bone marrow cells were injected into lethally irradiated mice. The numbers of CFUs found in control and salicylate-treated mice are shown in Table 3. No significant inhibition was seen. Mouse CSF production in response to intravenous injection of 5 pg E coli endotoxin was assayed in control and salicylate-treated mice (Table 4). Elevation of serum CSF in control endotoxin-treated mice was less marked than previously reported (1 1); however Table 2. Effect
01Salicylale Pretreatment in Viuo on CSF Stimulation of Mouse Bone Marrow Cells in Viuo
Treatment* Control Salicylate
Colonies/lP Cellst
Cells/Shaft x lo-ei
I84 f 48 175 f 47
19.0 f 1.8 22.9 f 3.3
"Bone marrow cells were taken from mice that had been injected intraperitoneally with 5 mg sodium salicylate in 0.5 ml of 0.5% NaCl four times daily for 5 days. These cells were cultured for 7 days in soft agar media containing C S F in the absence of salicylate, and the number of colonies was enumerated as described previously. Blood levels in mice reached 37-38 mg% I hour after each of three sequential injections spaced 4 hours apart. That drug accumulation occurred suggested that sodium salicylate is rapidly eliminated. t Results are the means from 6 mice f SD.
61
SALICYLATE AND LEUKOCYTE PROLIFERATION
Table 3. Salicylate Effect on Spleen CFUs CFUs/Spleen f SD (m) Treatment* Control Salicylate
EXPI t
EXP2t
28 f 5 (8) 1 8 f 8 (10)
17f4(11) 1 5 f 3 (11)
EXP 3$ 20f6(9) 18 f 3 (9)
* Syngeneic bone marrow cells ( I @ ) were adoptively transferred into irradiated mice. Spleens were excised and assayed for CFUs on day 7. No colonies were seen on spleens of mice that did not receive injections of bone marrow cells. t (C57BI X CBA) F, mice were irradiated with 900 R in two 450 R doses 24 hours apart. Salicylate-treatment mice received 30 mg sodium salicylate per day in four equal intraperitoneal doses (Exp I ) or in three equal doses (Exp 2). Drug treatment started 2 hours before adoptive transfer and was continued for 4 days. $. Recipient BALB/c mice were irradiated with a single 510 R dose. Both donor and recipient mice were pretreated with sodium salicylate for 7 days prior to adoptive transfer. Treatment of recipient mice was continued for an additional 7 days until sacrifice. Each mouse received 20 mg sodium salicylate per day in 4 equal intraperitoneal injections. salicylate treatment did not suppress CSF production and indeed appeared to enhance significantly the serum levels. Salicylate also suppressed human granulocyte and macrophage colony formation induced by CSA in vitro (Table 5 ) . This response was somewhat more variable than that seen with inbred mice (Table 1). Overall, however, the inhibition appeared to be dose-dependent for any individual patient. N o inhibition occurred when bone marrow cells were preincubated with salicylate in vitro for 2-5 hours and then washed and plated over an underlayer containing CSA (Table 6). None of the differences between control groups and comparable groups preincubated with salicylate for either 2 or 5 hours was significant at P < 0.2 (student's two-tailed r test).
DISCUSSION It has been shown that salicylates inhibit some lymphocyte functions and further suggested that they may possess immunosuppressant activity (6,17). The results presented here show that concentrations of salicylate similar to those inhibiting lymphocyte transformation (8-48 mg%) also inhibit CSF-induced proliferation of mouse CFUc in vitro when the drug is present continuously in the culture medium (Table 1). CSF used to stimulate mouse cultures is a glycoprotein with .a molecular weight of approximately 20,000. It is highly species-specific with effective dose levels in culture of the M (11). There is considerable order of lo-" to indirect evidence that CSF may also act in vivo as a
regulator of granulopoiesis and monocyte production. Other factors are involved, however, and the exact role of CSF in granulopoiesis in vivo remains to be established. Salicylate also inhibits human CFUc response to CSA in vitro with a similar dose response relationship (Table 4). CSA from cultured human leukocytes is less well characterized than mouse CSF and is stimulatory to both mouse and human CFUc. Although it has been established that salicylates inhibit the proliferative response of CFUc to CSF (or CSA) in vitro, it should be emphasized that the precise nature and location of the salicylate effect remains to be identified. Salicylate concentrations, used to inhibit generation of granulocyte and macrophage colonies in vitro, are similar to those found in salicylate-treated rheumatoid arthritis patients, but direct comparison to in vivo plasma concentrations is clouded because of possible differences in drug protein binding. Salicylate is not appreciably bound to plasma proteins in the mouse; 7% bound at 5 mg% and 5% bound as total plasma level reached 15 mg% (17-19). This result is in contrast to that in man, where protein binding is 75% at salicylate concentrations of 5 mg%, and decreases to 50% at 50 mg%. Both mouse and human bone marrow culture media contain 15% fetal calf serum. Salicylate inhibition of colony formation was demonstrable only when the drug was present in the soft Table 4. Salicylate Effect on E coli Endotoxin-Induced Mouse CSF Production Treatment*
CSF Units/O. I of Serum f SDt
Control Salicylate
8 f 3 68 f 14
* Twenty-three BALB/c mice were split into 2 groups; 12 mice received 0.5% NaCl and 11 mice received 250 mg sodium salicylate per kg per injection. Mice were injected three times daily for 30 days. Drinking water was withheld during the day and mice were given distilled water or sodium salicylate (3 mg/ml) in distilled water to drink overnight. Salicylate-treated mice consumed an extra 6.57 f 1.32 (SD) mg/ mouse/day via this route. The volume of fluid consumed was not significantly different for the two groups. t Mice were bled 3 hours after intravenous injection of 5 pg E coli endotoxin (DIFCO). Pooled blood samples were allowed to clot, and serum was removed and dialyzed against 0.02% sodium azide in distilled water for 24 hours and then against two changes of distilled water for an additional 48 hours. The serum was removed from the dialysis sacks, sterilized by millipore filtration, and assayed for colony stimulating activity with 7.5 X 10' C57B1/6 bone marrow cells. Serum was titrated in quadruplicate cultures over the range 1 : 1 to I :64 by the standard technique of Metcalf and Moore ( 1 I ) . CSF activity is expressed as the number of colonies stimulated in the linear portion of the dose response curve: 1 unit of CSF = 1 colony stimulated.
GABOUREL ET AL
62
Table 5. Effecf of Salicylate on Human Bone Marrow CSA Induced Colony Formation in Vitro
No. of Colonies/2 Patient
Diagnosis
1
Juvenile chronic myelogenous leukemia Agnogenic myeloid metaplasiaS Hodgkin's lymphoma Normal volunteer Neutropeniag Preleukemia
2 3 4 5 6
Nucleated Marrow Cells f SD* Salicylate 32 mg%t 48 mg%t
X l@
Control f SD
16 mg%t
251 rf: 123
268 f 34
101 f 15
4 0 f 15
2 f 2 35 f 10
251 17f8 6410 201 f 90 5 f 2
2.5 f 3 1715 2 f 1.4 171 f 2 7 2*0
4 9 f 10 3 0 f 10 49 f 6 280 f 9 1 2 f 10
-
270 f 41 13 f 7
*A
standard amount of CSA (100 pl) was added to each plate. Data are averages of colony counts from three to six plates. t Final salicylate concentration in the overlayer. $ C F U c obtained from spleen at time of splenectomy. 5 Marrow obtained during recovery phase.
agar culture and was not seen when bone marrow cells from salicylate-treated mice were cultured in the absence of drug (Table 2). Furthermore, no difference in total femoral shaft counts or morphology (bone marrow smears) could be detected. The fact that pretreatment of mice with large doses of sodium salicylate did not suppress colony formation in vitro might be explained by the reversibility of the salicylate-induced inhibition. Salicylate effects on lymphocytes in vitro have been shown to be reversible when the drug is removed by washing ( 6 ) .The present authors have also demonstrated that the effects on human bone marrow cells in vitro are similarly reversible (Table 5 ) . Thus washing of bone marrow cells in preparation for liquid agar culture may have removed or diluted any drug present as a result of in vivo treatment. The ability to reverse the salicylate effect by washing indicates that salicylate is not cytotoxic to human bone marrow cells and that the effects on granulopoieses are reversible. Chronic therapy with high doses of sodium salicylate for 30 days did not inhibit CSF production in response to intravenous injection of E cofi endotoxin, and indeed it appeared to enhance the response (Table 4). In this context Kurland and Moore (20,21) have observed that activated mouse peritoneal macrophages are a potent source of both CSF and prostaglandins of the E series. The latter are very potent inhibitors of CFUc in vitro, and physiologic concentrations of PGE, may serve to limit macrophage production of CSF in response to such activating agents as endotoxin. Because indomethacin treatment inhibits prostaglandin synthesis and enhances in vivo and in vitro CSF production (21 ), it is possible that the increased CSF production in sa-
licylate-treated mice is due to a similar mechanism. Kurland and Moore ( 2 2 ) have also shown that E-type prostaglandins directly inhibit the proliferation of committed granulocyte macrophage progenitor cells in vitro and that the inhibition can be overridden by increased concentrations of CSF. Because E-type prostaglandins have been demonstrated to suppress directly CFUc proliferation and to interfere with CSF production, it is unlikely that salicylate inhibition of prostaglandin synthetase could account for the suppressive salicylate effects described here. It is also interesting that acetylsalicylic acid (aspirin) and sodium salicylate are equally effective against experimental inflammation and in the treatment of rheumatoid arthritis (23), even though sodium salicylate has a much weaker inhibitory effect on prostaglandin synthesis in vitro (24). Furthermore, Smith et af (25) have shown that aspirin but not sodium salicylate caused a Table 6. Reversibility of Salicylate Inhibition of Colony Formation in Vitro afier Cell Washing Treatment Control (2 h r )
Amount of CSA Added 50 100
Salicylate (2 hr)
Salicylate (5 hr)
200 50 I00 200 50 I00 200
Colonies/2 X lo1 nucleated BM cells* 42 f 21 52 f 27 6 4 f 15 58 rf: 6 58 f 6 I21 f 55 76 f 31 40 f 6 81 f: 25
* Data are averages of colony counts for three plates
rf:
SD.
SALICYLATE AND LEUKOCYTE PROLIFERATION
significant reduction in the potentiation of carrageenininduced paw edema when arachidonic acid was given concurrently. Therefore the antiinflammatory effects of sodium salicylate may be mediated by a mechanism other than prostaglandin synthetase inhibiton. Because aspirin is rapidly converted to salicylic acid in vivo (26), it may be that it exerts part of its antiinflammatory activity by a second mechanism that is mediated by salicylate anion and does not involve inhibition of prostaglandin synthesis. Salicylate effects on the ability of certain adoptively transferred primitive hemopoietic stem cells to form macroscopically visible colonies of differentiating erythroid, granulocytic, or megakaryocytic cells in spleens of lethally irradiated mice were also investigated. N o significant effect on the number of CFUs per spleen could be detected when mice were pretreated with sodium salicylate prior to adoptive transfer and treatment continued during the period of in vivo culture (Table 3). It should be recognized, however, that in vivo CFUs are pluripotent stem cells, whereas the in vitro colony assay detects committed granulocyte monocyte progenitor cells (1 1 ). The lack of detectable effects on CFUs proliferation in the intact mouse may reflect the authors’ inability to maintain relatively constant high blood levels. No salicylate accumulation was found in mouse blood after three sequential intraperitoneal injections of 5 mg of sodium salicylate spaced 4 hours apart (Table 2 legend), a result suggesting almost complete elimination of an injected dose in the 4-hour period. In contrast, salicylate accumulation occurs in man during continuous therapy. When aspirin is given every 8 hours, plateau drug concerltrations are reached after 6 to 9 doses of 500 mg each or after 20 doses of 1000 mg each (27,28). Thus it is likely that high blood levels are better maintained in humans than in mice. It is unclear, however, whether chronic high-dose salicylate therapy results in suppression of granulopoiesis in man. In summary, salicylate is suppressive to CSF- to CSA-induced granulocyte and macrophage proliferation in agar. It is unlikely that these effect? are mediated through inhibition of prostaglandin synthetase. As yet the authors have been unable to demonstrate such suppression in vivo, and further studies in vivo are necessary to substantiate the possible clinical relevance of the in vitro effects described here.
ACKNOWLEDGMENT The authors are grateful to Ms. J. Yates for expert technical assistance.
63
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