Increased resistance to Listeria monocytogenes following subchronic cyclophosphamide exposure: Relationship to altered bone marrow function

CELLULAR

IMMUNOLOGY

Increased Subchronic

65, 13 1- 14 1 ( 198 1)

Resistance

to Listeria

Cyclophosphamide Altered Bone

Monocytogenes

following

Exposure: Relationship Marrow Function’

to

I. LUSTER,* GARY A. BOORMAN,? JACK $. DEAN,? LELA D. LAWSON,* RALPH E. WILsoN,t LLOYD D. LAuER,t ROBERT W. LUEBKE,* JOHN RADER,~AND LUCILLA CAMPBELL? MICHAEL

*Laboratory of Environmental Chemistry and tNationa1 Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received June 23. 1981: accepted September 24, 1981 Mice administered multiple doses of cyclophosphamide demonstrated a marked resistance to infection with the bacterium, Listeria monocytogenes. In contrast, acute exposure rendered mice more susceptible to infection than untreated controls. Resistance to infection with Listeria, a facultative intracellular organism, is thought to be dependent upon normal antimicrobial activity early after infection and subsequently through generation of primed T cells. Examination of various macrophage and immune functions, however, failed to demonstrate a significant difference between the two cyclophosphamide-treated groups although both groups were immunosuppressed when compared to untreated controls. Adoptive transfer studies into X-irradiated recipients revealed that repopulation with bone marrow cells from subchronic but not acute cyclophosphamide-treated mice, restored resistance. Furthermore, the numbers of granulocyte/macrophage progenitor cells in the bone marrow were elevated in subchronically treated mice but not acute or unexposed controls. These data suggest the selection of a granulocyte/macrophage progenitor cell possessing a high degree of antilisterial activity following subchronic cyclopbosphamide treatment. The effects induced by this exposure regimen are probably related to the enrichment of this cell population resulting from the cell cycle stage specific activity of the drug.

INTRODUCTION Increased resistance to infection by Listeria monocytogenes, a facultative intracellular microorganism, occurs in nude mice through a T-cell-independent accessory mechanism (1) and C57Bl mice through an autosomal, dominant non-H-2-linked gene (2). Increased resistance also occurs extragenically in mice which have been starved (3), splenectomized (4), pretreated with kinins (5), or treated with macrophage activators (6, 7). On the other hand, decreased resistance to Listeria infection occurs in mice following treatment with various immunotoxicants such as X irradiation (8), cytoreductive drugs including some alkylating agents, folic acid antagonists, antimitotic agents, and purine analogs (9), or macrophage toxins such ’ NTP No. 81-21. 131 OOOS-8749/81/170131-11/$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

132

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ET AL.

as carrageenen (8). Early studies established that resistance to Listeria is dependent upon mononuclear phagocytes activated by non-T-cell mechanisms early in the infectious phase (l-2 days) and by specific cell-mediated immunity of the delayed hypersensitivity type later in the infection ( 10). However, recent studies have implied that increased or decreased resistance to Listeria may be more complicated than previously suggested and are not only affected by genetic factors (2) but also by aging (1 l), suppressor T cells (12), precommitted T cells (i.e., Lyt 1,2,3) ( 13), and bone marrow cells (14). This report describes the differential affects resulting from acute and subchronic cyclophosphamide (CY) exposure on Listeria resistance and the relationship of this phenomenon to bone marrow functions. The effects of varying the dosage regimen on macrophage functions, immune tests and natural killer (NK) cell activity were also examined to determine whether this could account for the differential effects on host susceptibility. MATERIALS

AND

METHODS

Animals and dosing regimens. Specific pathogen-free female B&F1 (C57Bl/ 6N X C3H) mice weighing 18-22 g were obtained through NIH production contracts (Charles River, Portage, Mich.) C57B1/6 mice were obtained from Jackson Laboratories (Bar Harbor, Maine). The mice were 6-8 weeks of age, housed 10 per cage and allowed free access to food and water. CY was obtained from Mead Johnson and Company (Evansville, Ind.) and dissolved in sterile saline immediately prior to use. Mice were injected interperitoneally (ip) with either a single dose (acute) of CY (180 mg/kg) or multiple doses (subchronic) consisting of 36 mg/ kg per day for 5 consecutive days (total of 180 mg/kg) in 0.1 ml of saline. Control mice received saline. Animals were used 3 days following the last injection in various immunological, bone marrow, and host susceptibility assays. Listeria susceptibility assays. The bacterium, L. monocytogenes (Strain L242/ 73 type 4B) from a naturally infected mouse was used from the same frozen stock throughout the study. Organisms were grown overnight at 37°C in Trypticase soy broth. The number of bacteria in the broth, taken in a logarithmic phase culture, were quantitated turbidimetrically in a Beckman DB-G spectrophotometer at 540 nm and dilutions of the stock prepared in Hank’s balanced salt solution (HBSS). The number of viable bacteria in all dilutions used for animal inoculations were confirmed by conventional pour plate count procedures in Trypticase soy agar. In survival studies, groups of 10 mice received 0.5 ml of a selected dilution intravenously (iv), and were then monitored daily for 14 days or until death. Preliminary experiments had indicated that iv administration of about 1 X lo6 viable organisms resulted in an LD,, within 14 days. Bacterial enumeration in spleens were conducted on groups of five mice following a nonlethal iv infection (2 X lo4 organisms) as described previously (9). Bacterial counts were determined with an automated Artek counter (Model 880, Artek Corp., Farmingdale, N.Y.). The data on bacterial enumeration in the spleens are expressed as geometric means of viable counts based upon logarithmic scale. Cell preparations. Spleen cells were aseptically prepared as described previously ( 15). T-Cell enrichment was accomplished by passage of cells through columns

CYCLOPHOSPHAMIDE

AND Lisferia

RESISTANCE

133

containing glass wool followed by nylon wool as described by Trizio and Cudkowicz (16). Over 90% of the nucleated cells from the column passage stained with antiThy 1.2 (FITC-conjugated anti-mouse brain (C3H) antiserum; Litton Bionetics, Kensington, Md.). Macrophages were obtained from noninduced (resident) peritoneal cells through adherence to microexudate flasks according to the method of Mantovani er al. (17). All cell preparations contained greater than 95% viable cells as determined by trypan blue exclusion with over 95% stainable for nonspecific esterase. Adoptive transfer of listerial production. Recipient mice received 600 rad of total body irradiation at 40 rad/min using a Philips MG301 Model X-ray machine. Twenty-four hours later recipients were injected iv with either spleen cells, Tenriched spleen cells, adherent resident peritoneal cells, or bone marrow cells from either normal, acute, or subchronic CY-exposed mice. Mice received 4 X IO4 Listeria organisms 3 days after cell transfer and mortality was determined over a 14day period. In other experiments, bacterial enumeration was performed in spleens of irradiated mice reconstituted with bone marrow cells and challenged 5 days later with 2 X lo4 viable Listeria. Hematology and determination of splenic lymphocyte populations. Hematological profiles included erythrocyte counts, leukocyte counts, and differential. Splenic B and T cells were quantitated by the presence of surface immunoglobulins (SIg+) or Thy 1.2 antigen, respectively, by direct immunofluorescence employing monospecific antisera. The antisera utilized were FITC-conjugated goat anti-mouse polyvalent Ig (Microbiological Associates, Bethesda, Md.) absorbed with a 2% mixture of equal parts mouse liver powder and agarose and rabbit anti-mouse brain Thy 1.2 (C,H). Both antisera were used at a final dilution of 1:20 and a total of 200 nucleated cells were counted from each preparation using a Leitz fluorescent microscope (Model 20EB). Immunological tests. Delayed hypersensitivity responses to the T-dependent antigen, keyhole lymphet hemocyamin (KLH) were determined by a radiometric ear assay (18) based upon the original assay described by Lefford (19). Briefly, mice were sensitized by two subcutaneous injections of 0.1 mg KLH (Pacific Biomarine, Venice, Calif.) emulsified in incomplete Freund’s adjuvant 9 days apart and challenge intradermally with 0.03 mg of KLH in the pinna of the left ear. The right ear served as a control and was injected with saline. Twenty-four hours following challenge 6-mm ear plugs were taken, solubilized, and prepared for scintillation counting. The data are expressed as an index: DHR index =

cpm sensitive ear cpm control ear ’

In other mice antibody responses to SRBCs were determined by Cunningham’s modification of the Jerne plaque assay 4 days following iv immunization with 0.2 ml of a 5% solution of washed SRBCs (20). Macrophage (440) function assays. The microculture growth inhibition assay (GIA) for murine macrophage (i.e., cytostasis) activation was performed with pooled plastic adherent resident peritoneal cells using MBL-(2) leukemia target cells. The details of this procedure have been described (21). The percentage inhibition of target cell proliferation was calculated by

134

LUSTER

% growth inhibition

= 1-

ET AL.

cpm MBL(2) cpm MBL(2)

+ M4 from treated mice x 100. + M4 from control mice

For the phagocytosis assay, SRBCs were radiolabeled with NaS’CrO., (New England Nuclear, Boston, Mass.) and opsonized with anti-SRBC IgG (Cordis Laboratories, Miami, Fla.). Treated SRBCs were incubated with peritoneal cells in a 16-mm-well-diameter cluster dish (No. 3524, Costar Plastic, Inc., Cambridge, Mass.) as described by Snyderman et al. (22). The data are expressed as mean counts per minute of triplicate cultures as well as percentage change relative to the control group. Natural killer cell activity. Natural killer (NK) cell activity was determined in spleens of mice using YAC- 1 and RLd- 1 tumor cells as described by Herberman and Holden (23). The percentage cytolysis was determined by CPME-SR % cytolysis = 1 - CPMT-SR X 100, where CPME-SR is counts per minute in the experimental minus spontaneous release and CPMT-SR is counts per minute in total cells added (total release) minus spontaneous release. Bone marrow parameters. Marrow cells were aseptically collected from femurs and dispersed and the number of nucleated cell numbers determined with a Coulter Counter. Bone marrow granulocyte-macrophage progenitor cells (CFU-GM) were assayed using a method described by Bradley and Metcalf (24). Briefly, nucleated marrow cells were added to culture media ( lo5 cells/ml) containing 1.5% (w/v) methyl cellulose and 10% mouse lung-conditioned medium. One-milliliter aliquots of the cell suspension were placed in 35-mm petri dishes and allowed to incubate for 7 days at 37” in 7.5% CO, in air and the number of colonies per plate was determined using a stereomicroscope (Wild, Heerbrugg, Switzerland). Pleuripotent bone marrow progenitor cells, were quantitated by determining the number of colony-forming units in spleen (CFU-S) as described by Till and McCulloch (25). This was accomplished by iv injection of 5 X 1O4 nucleated bone marrow cells into 12-week-old irradiated (800 rad) B&F, recipients. Statistical analysis. The Mann-Whitney U test or x2 test was employed to assess the significance of treatment effects. RESULTS Susceptibility

of Mice to Listeria

Following

CY Exposure

Groups of 10 mice administered acute or subchronic doses of CY were given various concentrations of L. monocytogenes and survival was monitored daily over a 14-day period (Fig. 1). Administration of approximately 4 X lo6 Listeria resulted in an LDso in untreated B6C3F1 mice. A similar dose of Listeria given to mice pretreated with a single administration of CY ( 180 mg/kg) resulted in 100% mortality, while 100% survival occurred in mice exposed to CY subchronically (36 mg/ kg body wt daily for 5 days). Approximately 7 X 10’ viable organisms were required to produce an LDso in mice following subchronic CY exposure as compared to less than 5 X 10’ in mice that had received a single exposure. The time to death between control and CY-treated groups were similar in all experiments (data not shown).

CYCLOPHOSPHAMIDE

AND

-r

6.x105

6.x1@

NO.

LISTERIA

FIG. 1. Survival of mice infected with L. monocytogenes phamide (0), or acute cyclophosphamide (A) exposure. of 10 mice per group + SEM.

Organ Uptake

of Listeria

135

RESISTANCE

ee. 1, --\A ee. \ 0 48. I \ 28. \ 8. r

I le.

x SURVIVAL

Listeria

in CY-Treated

6.x~07

INJECTED

following Each value

saline (O), subchronic cyclophosrepresents mean triplicate values

Mice

Control and CY-treated mice were administered a nonlethal dose of Listeria (2 x 104) and the number of organisms in the spleen was determined over a 7-day period (Fig. 2). The number of bacteria in spleens of mice given a single injection of CY were similar to control values at 24 hr postinfection. However, after 24 hr a progressive bacterial growth occurred in the acutely exposed group eventually resulting in death. In contrast to acute exposure, subchronically treated mice revealed a persistent decrease in the number of organisms in the spleen when compared to controls which was comparable to survival data. Peripheral

Blood Values, Immune

Parameters,

and NK Activity

As summarized in Table 1, although CY treatment affected peripheral blood values, most immune parameters and NK activity, there were no marked differences between the two CY treatment groups to account for the difference in listerial resistance. Both acute and subchronic CY treatment-induced leukopenia although

1

2O

I

2 Days

3 4 post-infection

5

6

7

FIG. 2. Growth curve of L. monocyrogenes in spleens of normal mice (O), acute CY-treated (A), and subchronic CY-treated mice (0). Each point represents a mean of five mice. Different control values at P c 0.01 (**) and P < 0.05 (*) by Student’s z test.

mice from

136

LUSTER TABLE

ET AL. 1

The Effects of Varying CY Exposure Regimen on Peripheral Blood Values, Immmune Parameters and Natural Killer Cell (NK) Activity Treatment“ Parameter Peripheral blood WBC ( 10’/mm3) Lymphocytes (%) Monocytes (96) Spleen cell subpopulation’ I SIg positive I Thy-l.2 positive Effector DHR SRBC SRBC

function indexd PFC/ lo6 cells’ PFC/splcen (X10-‘)

Macrophage function Phagocytosis of “Cr-SRBCs (cpm) Cytostasis of MBL-2 tumor cells (cpm X 10’) NK activity ‘% Cytolysis with’ YAC-I tumor target RM-1 tumor target

None 1.4 2 0.4 822 3

1.5 2 0.2 41 31 2.9 -+ 0.2 1348 -c 151

Acute

Subchronic

r+_ 0.2' 81f 2 1.5 rt 0.2

1.7 + 0.2* 872 3 2.0 f. 0.3

2.3

23'

15b

156

156

30

1.2 + 0.1* 133 f 17” 49 f 5b

1387 +- 97 85.4 2 9.7

1073 ok 87 81.6 f 11.8

33.6

17.56 8.5'

2465

21.2

1.3 * 0.1* 170-c 40b 53-+

7b

1365 zk 79 81.6 + 7.7

9.4b 0.5*

a All values represent mean + SE of an average of eight mice. Separate animals were used for effector function tests since these assays required prior in viva sensitization. b Statistically different from untreated mice at P < 0.05. ’ Represents percent positive cells stained with FITC-conjugated antiserum against mouse Igs or mouse Thy 1.2. dDHRs were determined by administering 1 pCi/g body wt of [‘H]TdR (ip) into KLH sensitized mice 3 days following CY treatment followed 24 hr later by a challenge injection of KLH into the pinna of the left ear. The right ear was injected with saline and served as a control. The index was obtained by dividing cpm of the challenged ear by cpm of the control ear. ’ Immunizations were performed 1 day after CY treatment by iv injection of 0.2 ml of a 5% solution of washed SRBCs 4 days prior to sacrifice. f YAC-I and RM-I tumor cells were used at 1oO:l effector to target cell ratios in a 4-hr “Cr release assay.

this was not selective to any particular white cell population. The percentages of B and T lymphocytes as determined by the presence of SIg+ and Thy 1.2+ surface markers, respectively, were likewise decreased following either CY treatment, indicating an absolute as well as relative decrease in cells possessing these markers compared to controls. Effector cell function was also equally and markedly depressed following either CY exposure. Suppression of cell mediated immunity was evidenced in CY-exposed mice by a reduced ability to mount delayed hypersensitivity responses to KLH in sensitized mice with both treatment groups showing almost total anergy. Humoral immune responses appeared also to be severely impaired in both treatment groups as evidenced by a 90% reduction in antibody placque-forming-cell numbers following immunization with sheep red blood cells.

CYCLOPHOSPHAMIDE

AND

Lisreria

137

RESISTANCE

Macrophage

function was evaluated following CY exposure by quantitating in SRBCs and cytostasis coated, Wr-radiolabeled of MBL-2 leukemia target cells as measured in a growth inhibition assay. Phagocytic activity of adherent resident peritoneal cells from mice exposed to either CY exposure regimen was similar to control values. Similarly the ability of adherent peritoneal cells to inhibit the growth of MBL-2 leukemia target cells was not affected by CY treatment. Natural killer cell activity was depressed by well over 50% in both CY treatment groups compared to control values. However, again there was no significant difference between the two treatment groups to suggest the dichotomy in listerial resistance. vitro phagocytosis using antibody

Bone Marrow

Functions

To evaluate whether acute and subchronic CY exposure had differential affects on myelotoxicity the bone marrow cellularity, hematopoietic stem cell growth (CFU-S) and in vitro colony growth of granulocyte-macrophage progenitor cells (CFU-GM) were examined (Table 2). A marked bone marrow hypocellularity occurred following acute CY exposure and to a much lesser extent in subchronically exposed mice. The numbers of CFU-S were increased almost two-fold in both treated groups. Opposing effects were seen in the two CY treatment groups with respect to the number of CFU-GM progenitors which were markedly increased following subchronic treatment but similar to control values in acute exposed mice. Adoptive

Transfer

Studies

Adoptive cell transfer studies were conducted in an attempt to determine the cell type(s) responsible for the increased resistance to Listeria in mice subchronically exposed to CY (Table 3). In these experiments, spleen cells, T-cell-enriched spleen cells, adherent peritoneal cells, or bone marrow cells from normal or treated mice were transferred into X-irradiated recipients and subsequently challenged with Listeria. Approximately 4 X lo4 organisms resulted in 60% mortality in irradiated, nonreconstituted controls. Mice repopulated with spleen cells or T-cell-enriched spleen cells from subchronically treated animals were as susceptible to Listeria as irradiated mice that had received either saline or spleen cells from untreated mice. On the other hand, some protection may have been afforded following adoptive TABLE Bone Marrow

Cellularity Nucleated

Treatment None Subchronic Acute

and CFU

cells/femur (x10-6)

24.0 k 1.6 19.9 f o.9b 7.9 * 0.5’

2

Production

following

CY Administration”

CFU-S/ 5 X lo4 cells 8.3 f 13.5 * 15.2 k

a Values represent mean + SE of eight animals per group. ’ Significantly different from controls at P -c 0.05. ’ Significantly different from controls at P < 0.01.

0.5 0.7b 0.66

CFU-GM/femur (x1o-8) 186 f 20 258 f 18’ 176 + 34

138

LUSTER

ET AL.

TABLE Survival following Listeria Prior treatment of cell donors None None Subchronic Acute CY None Subchronic Acute CY Subchronic Subchronic

CY CY CY CY

3

monocytogenes Challenge in Irradiated Mice Repopulated with Cells from CY-Treated Mice”

Cell type transferred

Mortality/ No. tested

None Whole spleen cells Whole spleen cells Whole spleen cells Bone marrow cells Bone marrow cells Bone marrow cells T-Cell enriched spleens Adherent cells

13/21 9119 12119 9/17 o/10 o/10 8/10 5110 3/10

Percentage survival 38 53 31 47 100b 1006 20 50 70

’ Recipients received 600 rad on Day 0 and on Day 1 were injected iv with either saline, spleen cells (lo*), nylon wool-passed spleen cells (T-enriched; 5 X lo’), adherent RPCs (2 X lo’), or bone marrow cells (2.5 X 10’) from control or CY-treated mice. On Day 4 all mice received 4 X IO4 viable Listeria. This dose or organisms was not lethal (0 of 20) in untreated, nonirradiated control mice over a 14-day period. * Significantly different from control values at P e 0.05 by x2 analysis.

transfer of adherent peritoneal cells (not significant) while marked protection (P < 0.05) against Listeria infection occurred in irradiated mice following transfer of bone marrow cells from either control mice or subchronically CY-exposed mice, but not bone marrow cells from mice that were administered a single dose of CY. Table 4 depicts the growth of Listeria in spleens of irradiated mice reconstituted with bone marrow cells from normal or CY-treated mice. The differences between control and CY-treated mice were consistent with the earlier survival data. Mice repopulated with bone marrow cells obtained from subchronically exposed mice showed a significant decrease (P < 0.05) in the number of bacteria per spleen compared to mice reconstituted with bone marrow cells from normal or acute CYexposed mice. TABLE Enumeration of Listeria

Repopulated Yes

Yes Yes No

4

in Spleens of Irradiated Mice Reconstituted with Bone Marrow Cells from CY-Treated Mice” Treatment of cell donors

Ltteria/spleenb

None Subchronic CY Acute CY -

2.1 x IO5 3.5 x 10” 6.0 X lo5 >10*=

a Twenty-four hours following irradiation, mice intravenously received 2.5 X 10’ bone marrow cells, followed 5 days later with 2.0 X 10’ Listeria. Five days following bacterial challenge, mice were sacrificed and organisms/spleen determined. b Each value represents a mean of at least eight mice. ‘Significantly different from control values at P < 0.05.

CYCLOPHOSPHAMIDE

AND Listeriu RESISTANCE

139

DISCUSSION Studies initiated by Mackaness (10) demonstrated that resistance to Listeria infection during the first several days is dependent upon normal antimicrobial activity primarily through phagocytosis and killing by macrophages. Subsequently, resistance is dependent upon the induction of immunity through generation of primed T cells which activates the macrophage system resulting in increased microbicidal capacity. Consistent with this hypothesis, treatment of mice with substances which are toxic to macrophages or T lymphocytes will render animals more susceptible to infection (8, 9, 26). Conversely, immunopotentiators such as BCG or Corynebacterium parvum renders animals more resistant to infection (6, 7). The present studies clearly demonstrate that subchronic CY exposure renders mice resistant to Wisteria infection. On the other hand, acute CY exposure increases susceptibility to Listeria infection when compared to untreated controls and is consistent with earlier studies by Tripathy and Mackaness who also employed acute exposure (9). Listeria growth curves in spleens of mice following acute CY exposure indicate that rapid bacteria growth did not occur until several days following Listeria challenge. This is consistent with the view that CY preferentially affects lymphoid rather than myeloid cells (27) since Day 1 postinfection counts reflect nonspecific antimicrobial activity and Day 5 specific immunity. CY is a potent polyfunctional alkylating agent which exerts its cytotoxic action by crosslinking double-stranded DNA (28). The alkylating agent acts at the premitotic resting phase (G2) as well as the DNA-synthetic phase and is extremely toxic to precursors of the erythroid and lymphoid but less so for the myeloid series (27,29). In addition to affecting lymphoid precursor cells, CY may effect lymphoid cells at more advanced stages of differentiation including committed lymphocytes (30). CY-induced immunotoxicity has been reported to result in a spectrum of immune dysfunction ranging from a simple depletion of lymphocytes (30) to a functional impairment of the remaining lymphocytes (3 1). In these respects, both acute and subchronic CY-treated mice resulted in lymphoid depletion, suppressed effector cell functions and intact macrophage functions which is consistent with the above-described published reports. Since this immunological profile occurred as a result of acute CY treatment as well as subchronic exposure, it would appear that the enhanced resistance in the latter group is not a result of alterations in established mechanisms of Listeria resistance. Recently, Kaufman et al. (13) demonstrated that the Lyt 1,2,3+ T cell and not the more mature Lyt 1 or Ly 2,3 populations may be involved in protection against Listeria infection as well as delayed-type hypersensitivity to listerial antigens. CY has been shown to have selective effects on T-cell subclasses, having been reported, under certain dosage regimens to spare Lyt l-,2,3+-bearing lymphocytes (32). However, lymphocytotoxicity testing employing Lyt-specific antisera did not show this to be the case since both subchronic and acute CY exposure caused a similar effect on Lyt cell populations (unpublished data). Both exposure regimens also depressed natural killer cell activity indicating a lack of relationship with altered Listeria susceptibility. The two cyclophosphamide exposure regimens induced differential effects on bone marrow function. Marked hypocellularity occurred following acute CY exposure and to a lesser extent following subchronic exposure. In contrast to cellu-

140

LUSTER

ET AL.

larity, the number of CFU-S were enhanced in both subchronic and particularly acutely exposed mice, while the number of CFU-GM were markedly elevated following subchronic exposure. Earlier studies have demonstrated that treatment with cell cycle stage-specific (CCSS) drugs, like CY, has a selective cytoreductive action on the bone marrow (27). Following administration of these agents is an initial decrease in the number of CFU-S and CFU-GM followed later by increases in both primary and secondary hemopoietic organs (33, 34). The mechanisms responsible for this “repletion/depletion” phenomenon following treatment with CCSS agents are not clearly defined but vary depending upon the degree of cell kill, available space in secondary hemopoietic organs, and dosage regimens (35). In this respect, Buhles and Shifrine (36) reported that CFU-GM production in the bone marrow decreases immediately following an acute CY dose (250 mg/kg) which persists for days returning to normal levels by Day 4. In contrast, when two doses of CY were administered (250 mg/kg followed by 100 mg/kg 3 days later), while bone marrow hypocellularity also occurred, there was a 20-fold greater than normal proportion of CFU-GM in the bone marrow. In order to determine whether the differential affects on bone marrow functions induced by the two CY dosage regimens were related to listerial resistance, adoptive cell transfer studies were undertaken. Although survival data of this type should be interpreted with caution, it appears reasonable to assume that transfer of bone marrow cells from either normal or subchronically treated mice into irradiated recipients resulted in the ability of these animals to resist infection while transfer of bone marrow cells from acutely exposed mice failed to restore resistance. These data then support the theory that granulocyte-macrophage progenitor cells from the bone marrow can confer resistance against Listeria infection. This was further supported when spleens of irradiated mice repopulated with bone marrow cells from subchronically exposed mice were found to contain less bacterial organisms than spleens of irradiated mice repopulated with bone marrow cells from acute treated or control animals. Previous studies have shown that normal Listeria resistance can be restored in irradiated mice by bone marrow cell repopulation but not by transfer of peripheral lymphocytes or macrophages (14). Bennett and Baker (37) reported that mice treated with 300 mg/kg CY also demonstrated increased resistance to Listeria infection. They assumed this phenomenon was due to increased macrophage activation since treatment of the mice with silica abrogated the phenomenon. Previous studies in which treatment with macrophage activators increased resistance to Listeria infection demonstrated the protective role of nonspecifically activated macrophages in this infection (6, 7). The data in the present study would suggest that a granulocyte-macrophage precursor cell in the bone marrow, which coincidentally is activated or selectively increased by subchronic CY treatment, is also capable of conferring resistance against Listeria infection although macrophage activation or immunoenhancement does not occur. The origin and function of this cell does not coincide with natural killer cells which possess antitumor activity. That a preexisting mechanism of enhanced resistance may occur in nature in response to bacterial infection is suggested by the fact that nude mice maintained under germ-free conditions are more resistant to Listeria infection than their normal littermates although they lack mature T cells and do not possess an activated macrophage population (38). Macrophage activation occurs in nude mice held under conventional housing (1). In any case, it would appear that accessory mech-

CYCLOPHOSPHAMIDE

AND

Listeria

RESISTANCE

141

anisms, either in a quiescent or minimally active state, exist in normal mice that play a role in naturally minimizing bacterial growth early in infection until normal T-cell immunity has developed. ACKNOWLEDGMENT The authors

are grateful

to S. Wilkins

for preparing

the manuscript.

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