Protective Effect of Glucan Against Systemic Staphylococcusaureus Septicemia in Normal and Leukemic Mice
N. R. Di LUZIO* AND D. L. WILLIAMS
Department of Physiology, Tulane University School ofMedicine, New Orleans, Louisiana 70112
Received for publication 7 February 1978
The reticuloendothelial stimulant glucan, a beta-1,3-polyglucose component ofthe cell wall of Saccharomyces cerevisiae, was evaluated for its ability to modifyStaphylococcus aureus-induced lethality in normal and leukemic mice. In normalmice the intravenous injection of glucan (0.45 mg per mouse) 7 and 4 days priorto intravenous challenge with S. aureus (1.0 x 109) resulted in a significantlyincreased survival. Histological examination of the kidneys revealed that glucansignificantly inhibited renal necrosis associated with systemic staphylococcaldiseases. Further studies indicated that glucan administration not only enhancedsurvival of leukemic mice, but also increased survival of leukemic mice withexperimentally induced staphylococcal septicemia. These data denote that glucanenhances nonspecific resistance to S. aureus sepsis, promotes survival duringleukemic episodes, and increases survival time of leukemic mice with experimen-tally induced staphylococcal infection.
Previous studies have demonstrated glucan, acomponent of the cell wall of Saccharomycescerevisiae, to be a potent stimulant of the retic-uloendothelial system (6, 16). The intravenousadministration of glucan to mice results in hy-pertrophy of the major reticuloendothelial or-gans as well as a concomitant increase in acti-vation and proliferation of macrophages (18, 19,20). The hyperfunctional state of the reticulo-endothelial system induced by glucan is associ-ated with a marked enhancement of cellular andhumoral immunity (18, 19). Glucan has beendemonstrated to promote increased resistanceto tumor growth in an allogenic rat tumor (7)and in two syngenic mouse tumors (4). Prelimi-nary clinical studies have also indicated thatintralesional injection of glucan into human sub-cutaneous metastatic lesions results in the ac-cumulation of activated macrophages, prompttumor cell necrosis, and lesion regression (14,15).
In view of glucan's diverse immunological andantitumor activity, experiments were under-taken to evaluate whether glucan administrationwould modify experimentally induced bacterialsepsis in normal and leukemic mice. In recentyears, infection due to saprophytic or opportun-istically pathogenic microorganisms has becomea significant clinical problem (12). These infec-tions are particularly prevalent in patients whoare immunodeficient, due to the malignant stateor immunosuppressive therapy (11). The gram-positive opportunistic pathogen Staphylococcusaureus has been increasingly implicated as a
cause of secondary infection (11) and was, there-fore, chosen as the infectious model for thisstudy. The ability of glucan to enhance nonspe-cific protection against S. aureus septicemia innormal and leukemic mice was initially ascer-tained.
MATERIALS AND METHODSAnimals. AKR/J male mice were obtained from
Jackson Laboratory, Bar Harbor, Maine. The animalswere housed in plastic cages and were fed PurinaLaboratory Chow and water ad libitum.
Glucan. Glucan was prepared by a modification ofthe method of Hassid et al. (9). Dilutions were madein physiological saline (0.9% wt/vol) to provide 2.25mg/ml for intravenous injection. Isovolumetric salineserved as the control.
Bacteria. A clinical isolate of S. aureus was ob-tained from the Tulane Medical Center Hospital. Theorganism was subcultured in Trypticase soy broth for18 h at 370C in a shaking water bath (50 rpm). Identitywas verified by biochemical tests and purity by streakplating on blood agar. Isolated colonies were subcul-tured to Trypticase soy agar slants and maintained at40C. For each experiment, S. aureus was subculturedin Trypticase soy broth for 18 h at 370C in a shakingwater bath (50 rpm). The culture was centrifuged(2,000 x g) for 15 min, and the cell pellet was washedthree times with phosphate-buffered saline (0.9%wt/vol). Cell numbers were determined at 24 h intriplicate on Trypticase soy agar.Leukemia. AKR mice with lymphocytic leukemia
(BW 5147) were obtained from Jackson Laboratory.The leukemic tumor was maintained by the subcuta-neous injection of 106 tumor cells into 20-g AKR malemice. The cells for each experiment were prepared by
culturing AKR leukemic cells. Tumor cell suspensionswere subcultured into 25-cm2 Falcon tissue cultureflasks (Becton Dickinson Co., Oxnard, Calif.) contain-ing RPMI 1640 medium (Microbiological Associates,Walkersville, Md.) with 10% (vol/vol) fetal calf serum(KC Biological Inc., Lexena, Kan.), 2% (vol/vol) pen-icillin-streptomycin (Microbiological Associates), 0.1mg of gentamicin (Schering Corp., Kenilworth, N.J.)per ml, and 1% (vol/vol) amphotericin B (Grand IslandBiological Co., Grand Island, N.Y.). After incubationfor 4 days at 370C and 5.0% CO2 tension, the culturewas centrifuged (1,000 x g), and the cell pellet waswashed three times in RPMI 1640 without antibioticsor antifungal agents. Cells were counted on a hema-cytometer, and viability was determined by trypanblue exclusion. Dilutions were made in RPMI 1640with no antibiotics or amphotericin B.
Experimental procedures. In the initial bacterialresponse study, groups of mice were injected eitherwith glucan (1.0 mg per mouse) or isovolumetric salineon days 7 and 4 prior to challenge with 1.0 ± 0.25 x109 viable S. aureus.
In secondary infection studies, AKR lymphocyticleukemic cells (105) were injected intravenously intoall mice on day 0. Groups of mice were given glucan(0.45 mg per mouse) or saline intravenously on day 0,1, 2, and 5 and thereafter on alternate days up to day27. Peripheral leukocyte counts were employed tomonitor the onset of lymphocytic leukemia. On day13, one-half of the populations of each group werechallenged intravenously with 1.0 ± 0.25 x 109 S.aureus. All groups were monitored daily for survival.
Histology. Samples of liver, lung, spleen, kidney,and brain were taken on day 12 from glucan-pretreatedand saline control animals which were injected with S.aureus. All samples were fixed in 10% (vol/vol) For-malin. The sections were stained with hematoxylin-eosin.
Statistics. Statistical comparisons between groupswere performed employing Student's t test. A value ofP < 0.05 was considered significant. Statistical analy-ses of survival curves were based on chi-square with
ac
4
be
E
4
*so
50* - s-- - t~~~~~~~~I
a I
. |~~~~~~~~~~2c '\
a ~~~~- _I%-%1
o 2 6
1.+0.519 i
one degree of freedom. A chi-square value representing95% confidence level was considered significant. Allchi-square values represent a comparison between atreatment group and its appropriate control.
RESULTSEffect of glucan on susceptibility of mice
to S. aureus. To evaluate the protective effectof glucan against systemic staphylococcal infec-tion, glucan was administered intravenously 7and 4 days prior to intravenous challenge withS. aureus. At a challenge dose of 109 S. aureus,a 30% mortality was noted by day 1 in the salinecontrol group (Fig. 1). In contrast, only 3% mor-tality was observed in the glucan-pretreatmentgroup in an equivalent time period. The mediansurvival time for the saline control group wasapproximately 1.6 days as compared to 14 daysfor the glucan-pretreatment group. A 100% mor-tality was observed at 14 days in the salinecontrol group, at which time 60% of the glucan-pretreated mice were still alive. The glucan-pre-treatment group did not show 100% mortalityuntil day 26.Influence of glucan on the development
of leukemia. Systemic lymphocytic leukemiawas induced by intravenous administration of105 cultured AKR leukemic cells. Peripheral leu-kocyte counts were employed to monitor theonset of the malignant episode (Fig. 2). Within8 days, the saline control group showed a leu-kocyte count of approximately 12,000/mm3 (P< 0.01) (Fig. 2). In contrast, the glucan-treatedgroup did not show a comparable leukocytecount until day 13. By day 16, the peripheralleukocyte count in the glucan group was approx-imately 14,000/mm3. In contrast, the saline con-trol group had a peripheral leukocyte count of
10 14 18 22 26DAYS
FIG. 1. Glucan (1.0 mgper mouse) enhanced survival of mice to intravenous challenge with 1.0 + 0.25 x 109viable S. aureus. Glucan (- - -) was administered intravenously 7 and 4 days prior to challenge with 1i' S.aureus. Isovolumetric saline (--- -) served as the control in S. aureus-injected mice. N = 46per group.
FIG. 2. Comparative alterations in blood leuko-cytes ofAKR mice injected with 10' lymphocytic leu-kemic cells and subsequently treated with intrave-nously administered glucan ( ) or saline(---). N = 15 per group.
25,000/mm3 (P < 0.01) in an equivalent timeperiod. Although both the saline control andglucan group showed an increase in peripheralleukocyte count, there was a marked disparityin the rate of increase.
Effect of glucan on systemic staphylococ-cal disease in leukemic mice. Glucan, admin-istered in the dose of 0.45 mg per mouse on days0, 1, 2, and 5 and thereafter on alternate days upto day 27, resulted in a modification of survivalpatterns of leukemic mice (Fig. 3) as well asleukemic mice challenged intravenously on day13 with 109 S. aureus (Fig. 4). By day 20, leu-kemic mice that received glucan manifested an80% survival. However, the saline control groupshowed only 50% survival in an equivalent timeperiod. At day 25, the glucan group showed 70%survival as compared to 20% in the saline controlgroup (Fig. 3).Leukemic mice that received glucan prior to
and following the administration of S. aureusmanifested a 90% survival on day 20. In contrast,the saline control mice showed only 10% survivalin an equivalent time period. The median sur-vival time in the control group was 15 days, incontrast to 21 days in the glucan-treated group.By day 25, the glucan group showed 30% sur-vival, while all of the saline control mice withlymphocytic leukemia and experimentally in-duced staphylococcal secondary infection hadsuccumbed by day 23 (Fig. 4). The effectivenessof glucan in enhancing survival of mice injectedwith lymphocytic leukemia (Fig. 3) was signifi-cantly reduced when the mice received a subse-quent injection of S. aureus (Fig. 4).Histological observations. Histological ex-
amination of glucan-pretreated and saline con-trol animals on day 12 following intravenousinjection of 109 S. aureus revealed marked path-ological changes in the kidneys. There was a
__ ,........... \..
. _F~~~~~~~~~~~~-"'I ,,,,,,,,,
\~~~~..........
0 14 16 18 20DAYS
22 24 30
FIG. 3. Effect ofglucan administration on survivalofAKR mice following administration of IO' syngeniclymphocytic leukemic cells. Glucan ( ) or saline(,,,,II ) was administered intravenously on days0, 1, 2, and 5 and thereafter on alternate days to day27. N = 15 per group.
too
100
60
24
A
Ie@eX@@@
--
\P
0 14 16 18 20 22 24 30DAYS
FIG. 4. Glucan-induced enhancement in survivalofAKR mice which received 105 lymphocytic leukemiccells on day 0 and a subsequent intravenous injectionof S. aureus (i0d) on day 13. Glucan ( ) or saline( I I I I ) was intravenously administered on days0, 1, 2, and 5 and on alternate days up to day 27. Theincreased susceptibility of leukemic mice to intrave-nous S. aureus can be ascertained by comparison ofthe mortality patterns of normal mice (Fig. 3). Theexperiments depicted in Fig. 3 and 4 were performedsimultaneously. N = 15 per group.
diffuse acute pyelonephritis in the renal cortexof saline control mice (Fig. 5). Extensive necrosisof renal parenchyma was observed as a conse-quence of abscess formation (Fig. 6). There wasalso a marked dilation of renal tubules, whichwere filled with inflammatory cellular elementsand necrotic debris. In contrast to the severedegenerative changes in control mice, the kid-neys of glucan-treated mice exhibited relativelyminimal pathological changes due to the acuteseptic episode (Fig. 7). The livers of glucan-treated mice manifested the typical dose-de-pendent granulomatous reaction characterizedby a predominant monocytic infiltrate (Fig. 8).
VOL. 20, 1978 ANTI-STAPHYLOCOCCAL ACTIVITY OF GLUCAN 807
04 %**
.AV _ b-. q -;e _W ' -A~k V . -'- ., -49- 1 .41 qi #" a .- s-On*,
FIG. 5. Kidney ofsaline control mouse on day 12, showing a diffuse acutepyelonephritis in the renal cortex,characterized by necrosis of renal parenchyma and a chronic inflammatory cell infiltrate. Early abscessformation was observed along with a marked dilation ofrenal tubules filled with inflammatory elements andnecrotic debris (x200).
4r-0'&M 71-~~~~
lef
'U~~~~~~~l
.4,,1 U *
FIG. 6. Abscess formation in the kidney of a saline control animal on day 12. Extensive necrosis of renalparenchyma was observed as a consequence of abscess formation (x200).
FIG. 7. Kidney of a glucan-pretreated mouse on day 12 after S. aureus administration, showing minimalpathological changes along with a maintenance of renal integrity. (x200)
)X~#* wt A
.to
.* #w/r. 4
O-4 Wt,47
.4 5 # Z f
Ye* er^ z} ** *
*0 . ''ikv 44,S&'.t S
44
rifts "sX' .s 4. S +* %' 4
*E'w~ i;~''AS 4' ,;,,
FIG. 8. Liver of a glucan-pretreated mouse 12 days after S. aureus challenge, manifesting the typicalgranulomatous reaction characterized by a predominant monocytic infiltrate. (x450)
DISCUSSIONThe intravenous administration of glucan
prior to challenge with S. aureus was effectivein modifying morbidity and mortality due tosystemic staphylococcal infection. The apparentloss of protection in the later stages of the dis-ease may reflect a loss of protective efficacy dueto the reversible nature of the glucan-inducedhyperfunctional state (6, 10, 20). The currentstudies demonstrating the ability of glucan tomodify S. aureus infection agree with previousfindings that have shown protection in murineinfections with Sporotrichum schenckii (M. Ste-vens, P. Steven, J. A. Cook, H. Ichinose, and N.R. Di Luzio, J. Reticuloendothel. Soc. Prog.Abstr. 66a, 1976), Candida albicans (D. L. Wil-liams, J. A. Cook, E. 0. Hoffmann, and N. R. DiLuzio, RES J. Reticuloendothel. Soc., in press),Cryptococcus neoformans, and Mycobacteriumleprae (F. Lejeune, J. Delville, J. Gillet, M. Song,S. Stadsbaeder, and P. Jacques, Eur. J. Cancer,in press). These studies with bacterial and fungalpathogens, both opportunistic and overt, denotethat glucan induces a state of nonspecific hostresistance.The exact mechanism by which glucan pre-
vents sequelae to disseminated staphylococcaldisease has not been fully defined. However,earlier studies have demonstrated that glucanenhances both number and function of macro-phage populations (6). Additionally, Burgaletaand Golde have reported enhanced leukopoiesisfollowing glucan administration (5). They ob-served a twofold increase in peripheral leukocytecount along with an increase in the total mac-rophage content of the spleen, bone marrow, andperitoneal cavity of glucan-treated mice. Otherparameters of the immune system, such as com-plement (C3) (M. Glovsky, N. Di Luzio, and L.Ghekiere, J. Reticuloendothel. Soc. Prog. Abstr.,54a, 1976) and serum lysozyme (P. Kokoshis, D.L. Williams, J. A. Cook, and N. R. Di Luzio,Science, in press), have also been shown to in-crease due to glucan stimulation. This enhancedstate of innate defenses, promoted by glucan andprimarily mediated by phagocytes, may play animportant, if not crucial, role in protection of thehost against systemic staphylococcal disease.Based on earlier studies, mouse peritoneal
exudate macrophages have been shown to phag-ocytize and destroy non-encapsulated strains ofS. aureus (2). Biggar et al. have reported similardata using rabbit alveolar macrophages againstother gram-positive cocci, such as Streptococcusfaecalis and Streptococcus pneumoniae (3).While other components of the immune systemcannot be negated, the present observations, inconjunction with the above-cited studies, tend
to support the role of macrophages in host de-fense against S. aureus infections.
Studies with other immunostimulants, suchas BCG and Corynebacteriumparvum, have notshown an effect comparable to glucan on sur-vival of mice with experimentally induced staph-ylococcal septicemia. Adlam et al. reported noconsistent effect with intravenous or intraperi-toneal administration of C. parvum in mice thatwere subsequently challenged intravenouslywith S. aureus (1). In contrast, the data pre-sented here show that glucan provides signifi-cant protection with intravenous challenge dosesof S. aureus up to an order of magnitude higherthan the intraperitoneal challenge dose reportedby Adlam et al. (1). Sher and co-workers havereported that intraperitoneal pretreatment withBCG will modify survival of mice challengedwith 2.5 x 108 S. aureus (17). In comparison, ourdata demonstrated that intravenous administra-tion of glucan promotes significant protectionagainst intravenous challenge doses as high as10' S. aureus. Whether the failure of C. parvumto modify S. aureus lethality is due to the dif-ferential toxicity of glucan and C. parvum (13)remains to be established.The present data also demonstrated the pro-
tective efficacy of a glucan post-treatment regi-men against syngenic murine lymphocytic leu-kemia. Of equal importance, however, glucanwas effective in significantly modifying staphy-lococcal infection in leukemic mice. Secondaryinfections in leukemic children are of increasingclinical concern, and S. aureus is commonlyimplicated as one of the gram-positive organismsinitiating secondary infections in the leukemichost (12). In view of these observations, prophy-lactic treatment with glucan may be of potentialvalue in combating staphylococcal sepsis, partic-ularly as secondary infections in malignant epi-sodes.Based upon histological examination, the kid-
neys appear to be the most vulnerable organfollowing the intravenous administration of S.aureus. This observation is in agreement withthe data reported by Gorrill (8). Adlam et al.have suggested that the lack of protective effi-cacy of C. parvum administration against sys-temic S. aureus septicemia may be the result ofthe predilection of these microorganisms for thekidney (1). It was further postulated that thekidney is not affected by C. parvum-inducedlymphoreticular stimulation, and thus the pro-tective efficacy of C. parvum is lost followingsystemic infection with a nephrophilic microor-ganism. In contrast, our data suggest that glucanstimulation of the reticuloendothelial system iseffective in preventing degenerative changes in
the kidney. The protective efficacy of glucanadministration in S. aureus-treated mice is man-ifested by prolonged survival as well as minimalpathological changes in the kidneys of glucananimals as compared with controls.Our observations also denote that glucan in-
creases resistance to staphylococcal septicemiaand may confer duality of therapy by modifyingthe course of systemic leukemia while simulta-neously enhancing host resistance to S. aureus
infection.
ACKNOWLEDGMENTS
This study was supported by the Cancer Research InstituteofNew York, by the MECO Cancer Research Fund, by PublicHealth Service grant CA-13746 from the National CancerInstitute, and by the American Cancer Society.
LITERATURE CITED
1. Adlam, C., E. S. Broughton, and M. T. Scott. 1972.Enhanced resistance of mice to infection with bacteriafollowing pre-treatment with Corynebacteriumparvum.Nature (London) New Biol. 235:219-230.
2. Baughn, R. E., and P. F. Benventre. 1975. Phagocytosisand intracellular killing of Staphylococcus aureus bynormal mouse peritoneal macrophages. Infect. Immun.12:346-352.
3. Biggar, W. D., S. Buran, and B. Holmes. 1976. Bacte-ricidal mechanisms in rabbit alveolar macrophages: ev-
idence against peroxidase and hydrogen peroxide bac-tericidal mechanisms. Infect. Immun. 14:6-10.
4. Browder, W., E. Jones, R. McNamee, and N. R. DiLuzio. 1976. Inhibition of tumor growth by glucan, anonspecific immunostimulant. Surg. Forum 27:134-135.
5. Burgaleta, C., and D. W. Golde. 1977. Effect of glucanon granulopoiesis and macrophage genesis in mice. Can-cer Res. 37:1734-1742.
6. Di Luzio, N. R. 1976. Pharmacology of the reticuloendo-thelial system: accent on glucan, p. 412-421. In S. M.Reichard, M. R. Escobar, and H. Friedman (ed.), Thereticuloendothelial system in health and disease (func-tions and characteristics). Plenum Publishing Corp.,New York.
7. Di Luzio, N. R., E. 0. Hoffmann, J. A. Cook, W.Browder, and P. W. A. Mansell. 1977. Glucan-in-duced enhancement in host resistance to experimental
INFECT. IMMUN.
tumors, p. 495-499. In M. A. Chirigos (ed.), Control ofneoplasia by modulation of the immune system. RavenPress, New York.
8. Gorrill, R. H. 1958. The establishment of staphylococcalabscesses in the mouse kidney. Br. J. Exp. Pathol.39:203-212.
9. Hassid, W. Z., M. A. Joslyn, and R. M. McCready.1941. The molecular constitution of insoluble polysac-charide from yeast. J. Am. Chem. Soc. 63:295-298.
10. Hoffmann, E. O., N. R. Di Luzio, J. A. Cook, R.McNamee, and K. Armstrong. 1977. The morpho-logic effects of glucan in the liver, lung and spleen ofthe rat: light and electron microscopic studies. Lab.Invest. 36:340.
11. Hughes, W. T. 1971. Fatal infections in childhood leuke-mia. Am. J. Dis. Child. 122:283-287.
12. Hughes, W. T. 1977. Infections in the compromised host.Adv. Intern. Med. 22:73-96.
13. Lampert, I. A., P. D. Jones, T. E. Sadler, and J. E.Castro. 1977. Intravascular coagulation resulting fromintravenous injection of C. parvum in mice. Br. J.Cancer 36:17-22.
14. Mansell, P. W. A., N. R. Di Luzio, R. McNamee, G.Rowden, and J. W. Proctor. 1976. Recognition fac-tors and nonspecific macrophage activation in the treat-ment of neoplastic disease. Ann. N.Y. Acad. Sci.277:20-44.
15. Mansell, P. W. A., H. Ichinose, R. J. Reed, E. T.Krementz, R. McNamee, and N. R. Di Luzio. 1975.Macrophage mediated destruction of human malignantcells in vivo. J. Natl. Cancer Inst. 54:571-580.
16. Riggi, S. J., and N. R. Di Luzio. 1961. Identification ofa reticuloendothelial stimulating agent in zymosan. Am.J. Physiol. 200:297-300.
17. Sher, N. A., S. D. Chaparas, L. E. Greenberg, and S.Bernard. 1975. Effects of BCG, Corynebacterium par-vum, and methanol-extraction residue in the reductionof mortality from Staphylococcus aureus and Candidaalbicans infections in immunosuppressed mice. Infect.Immun. 12:1325-1330.
18. Wooles, W. R., and N. R. Di Luzio. 1962. Influence ofreticuloendothelial hyperfunction on bone marrowtransplantation. Am. J. Physiol. 203:404-408.
19. Wooles, W. R., and N. R. Di Luzio. 1963. Reticuloen-dothelial function and the immune response. Science142:1078-1080.
20. Wooles, W. R., and N. R. Di Luzio. 1964. The phagocyticand proliferative response of the reticuloendothelialsystem following glucan administration. RES J. Retic-uloendothel. Soc. 1:160-169.
