INFECTION AND IMMUNITY, Apr. 1978, p. 58-650019-9567/78/0020-0058$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 20, No. 1

Printed in U.S.A.

Increased Bone Marrow Production of Granulocytes andMononuclear Phagocytes Induced by Mycobacterial

Adjuvants: Improved Recovery of Leukopoiesis in Mice AfterCyclophosphamide Treatment

WILLIAM C. BUHLES, JR., AND MOSHE SHIFRINE*Radiobiology Laboratory, University of California, Davis, California 95616

Received for publication 10 November 1977

The effects of complete Freund adjuvant (CFA) or Mycobacterium bovis BCGon leukopoiesis and on leukopoietic recovery from cyclophosphamide treatmentin mice was studied. CFA injected subcutaneously or intraperitoneally resulted inincreased blood granulocyte and monocyte counts, increased numbers of bonemarrow granulocyte and mononuclear phagocyte progenitors, and increased he-matopoietic colony-stimulating factor in the serum. Furthermore, the quantitativecellular response within 24 h to an induced sterile intraperitoneal inflammation(thioglycolate) was augmented by subcutaneous CFA. In mice given CFA sub-cutaneously, blood granulocyte counts, as well as the peritoneal granulocyte andmacrophage response to intraperitoneal thioglycolate, recovered more quicklythan did those of the controls after a 250-mg/kg dose of cyclophosphamide. CFA-treated mice consistently maintained blood granulocyte and monocyte counts 1.3-to 4-fold higher than those of the controls for 2 weeks while receiving 75 mg ofcyclophosphamide per kg every other day. Mice pretreated with CFA intraperi-toneally had higher numbers of bone marrow colony-forming units in culture andhigher levels of serum colony-stimulating factor after 250-mg/kg injections ofcyclophosphamide. Similarly, BCG resulted in increased bone marrow colony-forming units in culture, increased serum colony-stimulating factor, and a fasterreturn of the peritoneal inflammatory response after cyclophosphamide injection.These results show that mycobacterial adjuvants accelerate recovery of leuko-poietic functions after cyclophosphamide treatment and suggest a mechanismwhereby such adjuvants afford nonspecific protection against infection in immu-nosuppressed mice.

Two recent investigations have shown thatthe induction of a state of enhanced nonspecificprotection against infection by immunostimula-tion with adjuvants results in increased survivalof mice given cyclophosphamide and subse-quently challenged with bacteria or fungi. Sheret al. (12) reported that Mycobacterium bovisBacille Calmette-Guerin (BCG), methanol ex-traction residue of BCG, or killed Corynebacte-rium parvum increases survival rates of cyclo-phosphamide-treated mice infected with Staph-ylococcus aureus or Candida albicans. We re-cently reported (4) that two mycobacterial ad-juvants, complete Freund adjuvant (CFA) andBCG, increase the 50% lethal dose for micetreated with cyclophosphamide and subse-quently infected with Pseudomonas aeruginosaor S. aureus. We proposed that this protectiveeffect could be attributed to increased produc-tion of granulocytes and mononuclear phago-cytes (leukopoiesis) by the bone marrow after

58

cyclophosphamide treatment and/or increasednonspecific microbicidal activity of macro-phages. The present study was initiated to in-vestigate the possibility that hosts treated withadjuvants may have improved bone marrow pro-duction of phagocytically active cells during im-munosuppression.There is considerable precedent for this con-

cept. In 1957, Smith et al. (14) reported that themarked susceptibility of mice to infection afterirradiation could be reversed partially by theinjection of endotoxin before irradiation. Themice treated in this way had an improved sur-vival rate against induced infection with P.aeruginosa. They also had higher granulocytecounts, faster granulocyte mobilization, in-creased bone marrow cellularity, and highernumbers of hematopoietic stem cells in the bonemarrow than mice irradiated without receivingendotoxin (15-17). The studies concluded thatendotoxin hastened recovery of bone marrow

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and thus improved the chance of survival afterirradiation.Bone marrow production of granulocytes and

mononuclear phagocytes can be augmented byadjuvants other than endotoxin. McNeill (9)showed that CFA injected intraperitoneally(i.p.) into mice increased the number of granu-locyte and mononuclear phagocyte progenitorcells (colony-forming units in culture [CFU-C])in the spleen, blood, and marrow. It also in-creased blood granulocyte and monocyte countsand increased the levels of serum hematopoieticcolony-stimulating factor (CSF). C. parvum andBCG also have been shown to augment thenumbers of CFU-C in bone marrow (6, 18, 19).However, to our knowledge, the effect of theseadjuvants on recovery from myelosuppressionhas not been reported previously.

MATERIALS AND METHODSMice. Male C3H.SW inbred mice obtained from E.

Benjamini (University of California, Davis) and maleC3H/HeN mice obtained from Charles River (Wil-mington, Mass.) were from 8 to 16 weeks of age.Specific-pathogen-free Swiss-Webster mice were ob-tained from Hilltop Farms (Scottdale, Pa.) and were12 to 16 weeks of age. All mice were housed four to sixper cage and given mouse chow (Simonsen's Labora-tories, Gilroy, Calif.) and tap water ad libitum.Cyclophosphamide. Cyclophosphamide (Mead-

Johnson, Evansville, Ind.) was prepared to 20 mg/mlwith sterile, distilled water immediately before use andinjected subcutaneously (s.c.) at a dose of 75 or 250mg/kg. Mice were weighed individually.

Adjuvants. CFA (Calbiochem, La Jolla, Calif.) waswarmed to 20'C and shaken to insure suspension ofmycobacterial cells but was not emulsified. IncompleteFreund adjuvant (IFA) was prepared from the samelot of CFA mentioned above by filtering through a0.22-,um membrane (Millipore Corp., Bedford, Mass.).BCG (Phipps strain, TMC 1029) was obtained fromthe Trudeau Institute (Saranac Lake, N.Y.) and con-tained greater than 10' colony-forming units per ml.BCG was stored at -50°C, quickly thawed immedi-ately before use, and passed repeatedly through a 27-gauge needle before injection. Control mice receivedinjections of phosphate-buffered saline (PBS).

Collection and quantitation of cells. Leukocytecounts were performed in a hemacytometer on bloodcollected from the tip of the tail and diluted 1:20 in 1%HCL. Cover-slip smears of blood were stained withWright stain, and differential cell counts were madeon 100 to 200 consecutive cells. Peritoneal cells werecollected by peritoneal lavage and counted as de-scribed previously (3). Bone marrow cells were col-lected by dissecting one or both femurs of each mousefree from soft tissue, cutting the bone at the metaph-ysis, and flushing the marrow cavity with 1.0 ml ofPBS (with heparin) by using a 27-gauge needle. Cellswere held on ice, and clumps were dispersed by passingthe suspension five times through a 22-gauge and thena 27-gauge needle. Cells were quantitated by dilutingin Eagle minimum essential medium (Grand Island

Biological Co., Grand Island, N.Y.) containing 2% Zap-isoton (Coulter Electronics, Inc., Hialeah, Fla.) andcounting on a hemacytometer. The ability of mice tomobilize exudative cells at the site of an inflammationwas assessed by injecting 2.0 ml of sterile thioglycolatebroth (Baltimore Biological Laboratory, Cockeysville,Md.) i.p. 24 h before peritoneal wash-out.

Quantitation of granulocyte and mononuclearphagocyte progenitor cells (CFU-C). Growth ofhematopoietic colonies in vitro from bone marrowrequires the addition to the culture medium of a sourceof CSF (2). CSF was provided in the present study bymedium conditioned with mouse L-cells, which wereobtained from Carleton Stewart, Division of RadiationOncology, Washington University, School ofMedicine,St. Louis, Mo. L-cell-conditioned medium was pro-duced as described previously (3).To quantify CFU-C, we used the conventional agar

culture assay for granulocyte and mononuclear phag-ocyte colonies (2). Colonies were grown in conditionedtissue culture medium consisting of Eagle minimumessential medium supplemented with 10% fetal calfserum, 5% normal horse serum, 20% L-cell-conditionedmedium, 0.1 mmol of L-glutamine, 100 U of penicillin,100 pg of streptomycin, and 0.25 pg of Fungizone perml. (All constituents except L-cell-conditioned me-dium were obtained from Grand Island Biological Co.)The conditioned tissue culture medium also contained10% of a 3% solution of freshly boiled Seaplaque aga-rose (Marine Colloids, Rockland, Me.) held at 37°C.Bone marrow cells, after counting, dispersion, anddilution in Eagle minimum essential medium, wereadded to the conditioned tissue culture medium, and2.0 ml containing 10i nucleated cells was plated ineach 35-mm plastic tissue culture plate (Falcon Plas-tics, Oxnard, Calif.). The agarose was allowed to gel at20°C for 10 to 20 min, and then the cultures wereincubated at 37°C in a humidified, 5% CO2 atmos-phere. By convention, colonies were defined as clonesof greater than 50 cells and were counted on day 7 ofincubation by using a Nikon dissecting microscopewith substage, reflective lighting at a magnification ofX15 to x20.Assay for serum CSF. The techniques used for

assaying serum CSF were essentially those of Metcalf(10) and of Robinson et al. (11). Whole blood wascollected from mice under chloroform anesthesia bycardiac puncture, allowed to clot at 20°C for 1 to 2 h,ringed, and cooled at 40C. After centrifugation at 500x g, the sera were removed, filtered through a 0.22-pum membrane (Millex, Millipore Corp.), and assayedimmediately or frozen at.-20°C for no more than 10days before assay.Bone marrow cells were collected from one femur

of each of one to four normal C3H.SW male mice,pooled, and treated as described above for quantita-tion of CFU-C. Tissue culture medium consisted ofEagle minimum essential medium supplemented with10% fetal calf serum, 5% normal horse serum, L-gluta-mine, and antibiotics as for conditioned tissue culturemedium, 10% of the filtered test serum (or L-cell-conditioned medium as a positive control), and 10% ofa 3% solution of Seaplaque agarose at 370C. Bonemarrow cells were added to give 105 cells per 2.0 ml.Plastic tissue culture plates (35 mm) were seeded with

VOL. 20, 1978 59

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60 BUHLES AND SHIFRINE

2.0 ml and incubated as for the CFU-C assay. Plateswere counted on day 7, scoring colonies (>50 cells)and clusters (10 to 50 cells) separately.

Statistical analyses. Statistical comparisons weremade by the Wilcoxin rank-sum test.

RESULTSEffect of CFA on peripheral leukocyte

counts. C3H.SW male mice were injected s.c.with 0.1 ml of CFA or 0.1 ml of PBS, andleukocytes were followed for 21 days. Adjuvant-treated mice had increased granulocyte andmonocyte counts by day 7 (Fig. 1). Granulocyteand monocyte counts in CFA mice were 1.5- to2.5-fold greater than those of controls. A singles.c. dose of cyclophosphamide (250 mg/kg) re-sulted in depression of granulocyte and mono-cyte counts. The monocyte nadir occurred 3days after the cyclophosphamide injection, withno significant difference between CFA and con-trol mice. The granulocyte nadir occurred 4 daysafter treatment, and control counts were higherthan CFA counts on both days 3 and 4 post-cyclophosphamide injection (day 3: 550 ± 96[mean ± standard error] for control versus 88± 28/mm3 for CFA, P < 0.01; day 4: 17 ± 5 forcontrol versus 1/mm3 for CFA, P < 0.02). Fivedays after cyclophosphamide treatment, granu-locyte counts were increasing, and CFA micehad significantly higher values (444 ± 86 forCFA versus 43 ± 14/mm3 for controls, P < 0.01).CFA mice maintained higher counts through 11days post-cyclophosphamide injection. Mono-

CFA CY12

_.,2 CFA sc

DIII Ia.ICO :C..ONTROL4

CnE 2-

0~z 'o

-4 -2 0 2 4 6 8 10 12 14DAYS

FIG. 1. Peripheral blood granulocyte and mono-

cyte counts in mice injected s.c. with CFA (-) or PBS(0) and subsequently treated with 250 mg of cyclo-phosphamide (CY) per kg. Symbols represent valuesfor the mean standard error of seven mice per

group.

cyte counts in CFA mice were more variable butgenerally higher than those of controls. Bloodlymphocyte counts were comparable in bothgroups.To establish the effect of CFA during contin-

ued administration of a lower dose of cyclophos-phamide, Swiss-Webster female mice were in-jected s.c. with 0.1 ml of CFA or PBS, and 10days later were started s.c. on cyclophosphamideat 75 mg/kg every other day. Swiss-Websterfemale mice were used in this study to avoid themarked monocytosis that occurs in C3H miceduring chronic cyclophosphamide administra-tion (3). Both granulocyte and monocyte countswere consistently higher in CFA mice (Fig. 2),indicating that adjuvant treatment enhancedleukopoiesis for more than 10 days of myelo-suppression. Granulocyte counts in CFA micewere 1.5- to 4-fold higher than those in controls,and monocyte counts were 1.3- to 3.5-fold higher.Lymphocyte counts never varied significantly.Adjuvant effects on bone marrow CFU-C

and serum CSF. The temporal response of thenumbers of CFU-C in the marrow and the levelsof serum CSF was determined in mice injectedi.p. with 0.1 ml of CFA at 10, 20, or 30 days

4500

CFA CY4000 I il

3500 S

WEAS+ sc 9

3 = 3000

O 500 I500~~~~~~~~~

1500-

~~E000~~CFA sT,) kT

z~~~~~~S

-3 0 10 14 18 22 26DAYS

FIG. 2. Blood granulocyte and monocyte counts inmice injected s.c. with CFA (0) or PBS (0) andsubsequently given 75 mg of cyclophosphamide (CY)per kg every other day as shown by arrows. Symbolsrepresent values for the mean + standard error of sixmice per group.

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EFFECTS OF ADJUVANTS ON LEUKOPOIESIS

before assay. Marrow CFU-C and serum CSFwere maximal on day 10 after CFA treatmentand declined thereafter (Table 1). Therefore, inall subsequent studies, mice received adjuvants10 days before cyclophosphamide treatment orcollection of cells.The effect of CFA (0.1 ml) or BCG (2 x 107 to

6 x 107 colony-forming units) given i.p. or s.c. onmarrow CFU-C and serum CSF was evaluated10 days after the injections. The effect of IFAwas also evaluated as a control for the non-mycobacterial component of CFA. CFA andBCG given i.p. resulted in increased bone mar-row CFU-C (Table 2). CFA and BCG given s.c.induced only moderate elevation in CFU-C. Se-rum CSF was elevated significantly after i.p.

TABLE 1. Temporal changes in marrow CFU-C andserum CSF after i.p. injection of 0.1 ml ofCFADays CFU-C Serum CSFb'

from ad- Cells perjuvant femur Colonies Clusterstreat- (XlOy'~ Per 10' Per fe-pe 0 prW

ment to cells mur per 1 erlscells cellsassay

Control 8,450 0.49 4.14 0 010 8,550 1.74 14.88 10 7720 6,900 0.90 6.21 0 3130 8,800 0.92 8.10 0 3

a Mean of four mice pooled per group.Mean of four plates per group.Expressed as number of colonies (>50 cells) or clusters

(10 to 50 cells) induced per 105 normal bone marrow cellsplated.

injection of both adjuvants but to a lesser degreeor not at all by s.c. administration. (Because ofvariations in technique and in the sources ofnormal bone marrow, it is not possible to com-

pare serum CSF activity in one experiment withthat in another. Strict comparison should bereserved only for values within an experiment.)Absolute numbers of CFU-C per femur were

increased 1.8- to 5-fold over those of controls byadjuvants given i.p., indicating a significantlyenhanced capacity for cell production by themarrow. IFA had no effect on marrow CFU-Cand serum CSF values.We also compared the reduction and subse-

quent recovery of bone marrow CFU-C inducedby 250 mg of cyclophosphamide per kg in controlmice and in mice injected i.p. with CFA. CFAmice had increased bone marrow cellularity andCFU-C compared with the controls (PBS i.p.)on days 0, 1, and 2 (Table 3). The CFU-C nadiroccurred on day 1, when CFA mice had 3.6-foldmore CFU-C per femur than control mice. Therelative number of CFU-C appeared to reboundhigher, and then fall off faster, in CFA mice.Bone marrow cellularity reached a nadir on day3, and cellularity in CFA mice was reduced to a

level equal to or below that of the controls. Atall periods after cyclophosphamide injection, se-

rum CSF activity was significantly higher inCFA mice than in control mice.

Effects of adjuvants on peritoneal cellsand their response to an inflammatorystimulus. To test whether adjuvants would en-

TABLE 2. Changes in bone marrow cellularity, CFU-C, and serum CSF in mice injected withmycobacterial adjuvants

CFU-C Serum CSF'

Exptno.Treatmenta Cells/femurExpt no. Treatment Cells/r(X0)b Per 10:' cells' Per femur Colonies per Clusters per l0i0' cells cells

1 PBS i.p. 8,200 0.48 3.94 0 <1CFA s.c. 8,200 1.00 8.20 1 26CFA i.p. 10,500 1.85 19.43 51 NC"

2 PBS i.p. 6,425 0.99 6.36 0 0CFA s.c. 9,950 0.95 9.45 0 0CFA i.p. 10,950 1.61 17.63 17 NC

3 PBS i.p. 8,850 0.45 3.98 0 5BCG s.c. 8,625 0.65 5.61 0 4BCG i.p. 9,950 0.74 7.36 0 24

4 PBS i.p. 7,700 0.86 6.62 0 0BCG i.p. 9,075 1.56 14.16 0 8IFA i.p. 7,850 0.87 6.83 0 0

aPBS (0.1 ml), CFA (0.1 ml), BCG (2 x 107 to 6 x 107 colony-forming units), or IFA (0.1 ml).was injected 10days previously.

h Mean of four mice pooled per group.c Mean of four plates per group.d NC, Not counted.

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62 BUHLES AND SHIFRINE

hance the delivery of cells to a site of inflam-mation distant from the adjuvant administra-tion, BCG or CFA was given s.c., and the peri-toneal inflammatory response to a subsequenti.p. injection of thioglycolate broth was mea-

sured.Thioglycolate administration resulted in a

marked granulocyte and mononuclear phago-cyte accumulation within 24 h after injection(Table 4). Lymphocytes were not affected. CFAand BCG given s.c. 10 days earlier caused aslight but insignificant increase in the number of

macrophages and lymphocytes in the unstimu-lated peritoneal cavity. However, the mononu-clear phagocyte response and, to a lesser extent,the granulocyte response to thioglycolate wasgreater in CFA and BCG mice. Slightly higherlymphocyte counts persisted in adjuvant-treatedmice after thioglycolate injection. These datasuggest that adjuvants slightly enhanced theability to mount an inflammatory response at asite distant from adjuvant administration.The effect of adjuvants on the recovery of the

peritoneal inflammatory response after cyclo-

TABLE 3. Effect ofCFA i.p. on recovery of bone marrow CFU-C and serum CSF after injection of250 mg ofcyclophosphamide per kg

CFU-C Serum CSF"Treatment Days post- Cells per femurCYb (x0)' P 'cell Per femur Colonies per Clusters perPer io~~~~~~~~0cells iO' cells

PBS 0 7,075 0.43 3.04 0 <1CFA 9,075 0.83 7.53 0 23

PBS 1 2,400 0.28 0.67 0 2CFA 5,200 0.47 2.44 0 7

PBS 2 1,150 2.27 2.61 0 3CFA 1,625 2.35 3.82 <1 22

PBS 3 360 24.72 8.90 0 2CFA 290 36.31 10.53 4 58

PBS 4 1,050 17.84 18.73 0 7CFA 1,500 10.20 15.30 0 22a PBS or CFA (0.1 ml) given i.p. 10 days previously.'CY, Cyclophosphamide.e Mean of four mice pooled per group.d Mean of four plates per group.

TABLE 4. Peritoneal cell counts and the peritoneal inflammatory response to thioglycolate in micepretreated subcutaneously with adjuvants

Peritoneal washout cell count (mean x l:/ml)Treatment" Peritoneal stimula- No. of micetion Totalb Granulocytes Macrophages Lympho

cytesNone None 6 1,233 0 400 786None TG-24hc 6 4,988d 1,790" 2,315d 723PBS None 7 1,100 3 346 705CFA None 7 1,454d 0 457 947PBS TG-24h 18 4,649 2,783 1,184 568CFA TG-24h 18 6,174d 3,301 1,852d 884dPBS None 6 1,075 14 521 495BCG None 6 1,217 5 573 595PBS TG-24h 12 5,773 3,632 1,454 656BCG TG-24h 12 6,696 3,752 2,163 758

a CFA (0.1 ml) s.c., BCG (2 x 107 to 6 x 107 colony-forming units) s.c., or PBS (0.1 ml) s.c., 10 to 11 daysbefore cell collection.

' Total cell count may not equal sum of three cell types since other minor populations of cells (e.g., mastcells) are not shown.

e TG-24h, A 2.0-ml amount of thioglycolate broth given i.p. 24 h before cell collection."Significantly different from control by Wilcoxin test (P < 0.05).

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EFFECTS OF ADJUVANTS ON LEUKOPOIESIS

phosphamide treatment was tested on mice in-jected s.c. with CFA or PBS and 10 days laterinjected s.c. with 250 mg of cyclophosphamideper kg. On days 1 through 6 after cyclophospha-mide injection, 2.0 ml of thioglycolate was in-jected i.p., and the peritoneal cell response was

quantitated 24 h later. In CFA-treated mice, themacrophage response was enhanced on days 4and 5, and the granulocyte response was en-

hanced on days 5 and 6 (P < 0.05) (Fig. 3). Afterthis, the responses of the control animals tendedto overshoot those of the CFA-treated mice, butthese differences were not significant (P > 0.05).Lymphocyte counts were unaffected by CFA.The peritoneal inflammatory responses tendedto mimic the fall and recovery of the leukocytecounts after injection of cyclophosphamide; themononuclear phagocyte nadir occurred on day3, and the granulocyte nadir occurred on day 4.On day 4, CFA mice had a significantly lowergranulocyte response than did control mice.Because the most marked enhancement of

phagocytic cell responses by CFA after recoveryfrom cyclophosphamide occurred on day 5, weperformed four additional experiments to deter-mine the effect ofBCG or IFA on the peritonealinflammatory response on that day (Table 5).Some variation in the counts for saline-treatedcontrol mice occurred between experiments,probably because of differences in animal groups

and minor variations in timing of cyclophospha-mide and thioglycolate administration. BCGmarkedly improved the peritoneal inflammatoryresponse of both granulocytes and mononuclear

I0,00c

1,000 ".CFA sc

E

- dCONTROL

CFA0sc

1,000

E O~~~~~,CNTROL

1003U

0"7' 2 3 4 5 6 7

DAYS AFTER CYCLOPHOSPHAMIDE

FIG. 3. Peritoneal granulocyte and macrophageinflammatory responses to thioglycolate at varioustimes after cyclophosphamide (250 mg/kg) treatment.Mice received 0.1 ml of CFA (0) or PBS (0) s.c. at-10 days and were injected with cyclophosphamides.c. on day zero; thioglycolate was injected i.p. 24 hbefore cell collection. Symbols represent values forthe mean offive to six mice each.

TABLE 5. Peritoneal cell response to thioglycolate 5days after treatment with cyclophosphamide in mice

injected s.c. with mycobacterial adjuvants

Peritoneal cell response to thiogly-

Treat- colate (mean x 10'/ml)Expt no. menta Granulo- Macro- Lympho-

cytes phages cytes

1 PBS 30 2,157 150CFA 247b 4,803 152

2 PBS 90 2,684 508CFA 288A 4,649b 593

3 PBS 12 420 115BCG 202b 2,992b 364b

4 PBS 3 535 138BCG 479b 2,882b 459b

5 PBS 4 819 70IFA 12 1,007 164b

6 PBS 10 541 52IFA 16 737 88

- CFA (0.1 ml), IFA (0.1 ml), BCG (2 x 107 to 6 X

107 colony-forming units), or PBS (0.1 ml) was injecteds.c. at -10 days. Mice received 250 mg of cyclophos-phamide/kg s.c. on day zero and 2.0 ml of thioglycolatei.p. on day 4 after cyclophosphamide injection, andthe peritoneal count was performed on day 5.

bp< 0.05.

phagocytes, but IFA did not. There was also asignificantly greater number of lymphocytes inthe peritoneal washout in mice pretreated withBCG or IFA.

DISCUSSIONCFA given s.c. produced only a small, local-

ized, granulomatous lesion at the site of injectionand resulted in a surprising increase in leuko-poietic activity, essentially doubling the numberof granulocytes and monocytes in the blood andnearly doubling the number of bone marrowCFU-C. The i.p. injection of CFA, which re-sulted in a considerably greater degree of tissueinvolved in the granulomatous inflammatory re-sponse, had an even greater effect on CFU-C.Significantly increased levels of serum CSF wereobserved with CFA given i.p., but only one ex-periment showed enhanced CSF production bys.c. CFA, and it was at a considerably lower levelthan with i.p. injection. The sensitivity of theCSF assay technique may have been too low toconsistently detect serum CSF activity belowcertain critical levels, i.e., levels that might havemajor physiological effects in vivo.Bone marrow production of granulocytes and

mononuclear phagocytes, as represented byCFU-C per femur, was increased by CFA evenafter administration of cyclophosphamide. This

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64 BUHLES AND SHIFRINE

increase was consistent with the improved re-covery of leukocyte counts and the higher, morepersistent leukocyte counts in CFA mice treatedwith the drug. Serum CSF activity was alwayshigher in CFA-treated mice after injection ofcyclophosphamide. This finding supports thehypothesis that the improved bone marrow pro-duction is mediated by the action of CSF andthat agents which augment CSF production,such as endotoxin and adjuvants, allow im-proved recovery from a myelosuppressive insult.The kinetics of the recovery of granulocyte andmononuclear phagocyte production in adjuvant-treated versus control mice are similar to therecovery curves observed by Smith et al. (15, 16)when endotoxin was administered before irradia-tion. These similarities between endotoxin andadjuvants suggest a common physiologicalmechanism of enhanced leukopoietic recovery,most likely related to CSF.

Infection of mice with viable BCG resulted inleukopoietic enhancement similar to that in-duced by CFA treatment. BCG given i.p. in-creased bone marrow CFU-C by a factor of 1.8to 2.1 and slightly increased serum CSF levels.BCG did not appear to be quite as potent asCFA in its effect on the marrow, but comparisonof these effects was difficult because results wereobtained from different experiments. BCG in-duced only slightly higher numbers of peritonealgranulocytes and macrophages in the unstimu-lated, as well as the stimulated, peritoneal cav-ity. BCG was very active in augmenting therecovery of the peritoneal inflammatory re-sponse after cyclophosphamide injection. UnlikeCFA, this enhanced cell recovery included lym-phocytes as well. Apparently, the active com-ponent of CFA is the mycobacterial cell, sinceIFA neither stimulated leukopoiesis nor en-hanced peritoneal inflammatory responses.CSF may play a primary role in the enhanced

leukopoietic activity after adjuvant administra-tion, but the mechanism of adjuvant-inducedCSF production is still unknown. CSF can beextracted from a variety of mouse tissues (13),but in vitro production of CSF appears to belimited to mononuclear phagocytes (includingmonocytes and macrophages), lymphocytes, andfibroblasts (1, 8). Mononuclear phagocytes cul-tured in vitro produce CSF, which can be de-tected in the culture medium, and exposure ofthese cells to endotoxin increases the rate ofCSF production in a manner qualitatively simi-lar to the in vivo CSF response after endotoxinis administered (5, 7). Thus, endotoxin admin-istered in vivo may activate macrophages andresult in the release of increased amounts ofCSF, which subsequently enhances leukopoieticfunctions. Similarly, a specific, lymphocyte-me-

diated immune response to mycobacterial anti-gens may result in the production of lympho-kines that would cause macrophage activationand lead to the release of increased amounts ofCSF. Alternatively, mycobacterial cell compo-nents may directly activate macrophages or lym-phocytes to release CSF.The significantly improved delivery of granu-

locytes and macrophages to the primary site ofan inflammation in adjuvant-treated immuno-suppressed mice in the present study may ac-count for the increased survival rate of suchmice after an infectious challenge, as demon-strated in previous experiments (4, 12). We alsorecently reported that tissue macrophage num-bers vary less than the numbers of granulocytesor lymphocytes after treatment with cyclophos-phamide and that macrophages collected fromcyclophosphamide-treated mice appear to befunctionally normal (3). Thus, adjuvant-treated,immunosuppressed mice may also have aheightened level of macrophage activity that isrelatively cyclophosphamide resistant. The in-creased production and delivery of phagocyticcells by adjuvants, coupled with the survival offunctional macrophages, may represent twocomponents of nonspecific protection against in-fection during immunosuppression with cyclo-phosphamide.

ACKNOWLEDGMENTS

This research was supported by Public Health Servicegrants 5501RR05457 and 5TIGM0076212 from the GeneralResearch Support Branch, Division of Research Resources,National Institutes of Health and National Institute of Gen-eral Medical Sciences, respectively; by an American CancerSociety institution grant to the University of California, Davis;and by the Energy Research and Development Administra-tion.We are most thankful to Shiona MacKay for excellent

technical assistance.

LITERATURE CITED

1. Austin, P. E., E. A. McCulloch, and J. E. Till. 1971.Characterization of the factor in L-cell conditioned me-dium capable of stimulating colony formation by mousemarrow cells in culture. J. Cell. Physiol. 77:121-134.

2. Bradley, T. R., and D. Metcalf. 1966. The growth ofbone marrow cells in vitro. Aust. J. Exp. Biol. Med. Sci.44:287-300.

3. Buhles, W. C., and M. Shifrine. 1977. Effects of cyclo-phosphamide on macrophage numbers, functions, andprogenitor cells. J. Reticuloendothel. Soc. 21:285-297.

4. Buhles, W. C., and M. Shifrine. 1977. Adjuvant protec-tion against bacterial infection in granulocytopenicmice. J. Infect. Dis. 136:90-95.

5. Cline, M. J., B. Rothman, and D. W. Golde. 1974.Effect of endotoxin on the production of colony-stimu-lating factor by human monocytes and macrophages. J.Cell. Physiol. 84:193-196.

6. Dimitrov, N. V., S. Andre, G. Eliopoulos, and B.Halpern. 1975. Effect of Corynebacterium parvum onbone marrow cell culture. Proc. Soc. Exp. Biol. Med.148:440-442.

7. Eaves, A. C., and W. R. Bruce. 1974. In vitro productionof colony-stimulating activity. I. Exposure of mouse

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