INFECTION AND IMMUNITY, Jan. 1977, P. 26-33Copyright O 1977 American Society for Microbiology

Vol. 15, No. 1Printed in U.S.A.

Mitogenic Stimulation of Murine Spleen Cells: Relation toSusceptibility to Salmonella Infection

NICOLAS VON JENEY, EBERHARD GUNTHER, AND KLAUS JANN*Max-Planck-Institut fur Immunbiologie, D-78 Freiburg, Stubeweg 51, West Germany

Received for publication 1 June 1976

The screening of several inbred strains of mice suggested that the capacity oftheir spleen cells to respond to the mitogenic effect of lipopolysaccharide (LPS) ofgram-negative bacteria was correlated with their resistance to intraperitonealinfection with Salmonella typhimurium. An injection of LPS into mice causedchanges in the in vitro responsiveness of their spleen cells to the mitogeniceffects ofLPS and phytohemagglutinin. Pretreatment of mice with whole ultra-violet (UV)-killed bacteria led to a marked rise in the in vitro response of thespleen cells to UV-killed bacteria, but not to LPS or phytohemagglutinin. Thisenhanced response to UV-killed bacteria was not specific for the 0 antigens ofthe bacteria.

Salmonella typhimurium is a facultative in-tracellular pathogen of mice that causes a sys-temic infection (43). Overwhelming infectionresults in bacteremia and death; mild infectionsresult in a carrier state, with concomitant im-munity (13, 14, 22), or the bacteria may beeliminated by immune mechanisms of a cellu-lar nature. This is followed by a rather short-lived nonspecific immunity, but a prolongedspecific memory to the pathogen (6). Therefore,a central issue ofpathogenesis and resistance isthe interaction of the invading organism and itsproducts with the lymphocytes of the host.The cell wall of gram-negative bacteria con-

sists largely of a lipopolysaccharide (LPS)known as endotoxin (21). Its multiple effects onthe higher organisms have been extensivelystudied (15, 16). One of the properties ofLPS isits mitogenicity for B-lymphocytes (2, 3, 9, 29,34, 36). Lymphocytes of different inbred strainsofmice differ in their capacity to respond to themitogenic effect of LPS; C3H/HeJ mice are lowresponders (8, 10, 33, 39, 40). This, however,seems to depend on the method usd for theextraction of LPS from the bacteria (37).

Since it is known that inbred strains of micealso may differ in their susceptibility to infec-tion with S. typhimurium (11, 30, 32, 41), wewished to determine whether there is a correla-tion between the responsiveness of mousespleen cells to the in vitro mitogenicity of LPSand whole bacteria and the susceptibility of themice to this infection.The in vivo interaction of lymphocytes with

LPS and whole bacteria may alter their in vitroresponsiveness to mitogens. We thus studiedthe proliferative responses of the spleen cells of

three selected strains of inbred mice to wholebacteria, LPS, and phytohemagglutinin (PHA)after the injection of LPS or whole bacteria.

MATERIALS AND METHODS

Mouse strains. Mice of the inbred strains C57B1/6, C3Hf, CBA/H, and DBA/2 were bred under spe-cific pathogen-free conditions, and AKR/J, Bl0.D2/n, C3H/DiSn, and C3H.SWSn mice as well as(C57Bl/6 x C3Hf)Fl hybrids were bred under con-ventional conditions in this institute. The C3H/HeJmice were purchased from the Jackson Laborato-ries, Bar Harbor, Maine. Male mice, 12 weeks old,were usually employed in all experiments, althoughno differences were found between the reactions ofmales and females. All mice were kept under con-ventional conditions for 2 to 4 weeks prior to theexperiments. Herilan Han MR3 pellets (H. Eggers-mann KG, 326 Rinteln/Weser, West Germany) andwater were given ad libitum.

Bacterial strains. The following bacterial strainswere used: S. typhimurium F1826 (lysotype LT20, 0antigenic factors 1, 4, 5, 12, 123); S. typhimuriumF1826a, a smooth variant of strain F1826 of reducedvirulence for mice, but with the same 0 antigenicfactors; and Escherichia coli O111:K58(B):H-(F2125).

Bacterial growth media. Stock cultures of thebacterial strains were maintained on egg agar (18).For the counting of colonies, agar plates, accordingto Loeb and Zinder (20), were used. Standard-Inutrient broth (E. Merck AG, Darmstadt, WestGermany) was used for growing bacterial suspen-sions.

Tissure culture media. Eagle medium with theDulbecco modification (Grand Island Biological Co.,Grand Island, N. Y.) supplemented with streptomy-cin (100 ,ug/ml) and penicillin (100 U/ml) was used.For the preparation and washing of spleen cells themedium was buffered with 20 mM HEPES (N-2-

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hydroxyethylpiperazine-N'-2-ethanesulfonic acid)(C. Roth, 75 Karlsruhe, West Germany) (Eagle-HEPES). For the culturing of cells the medium wasbuffered with sodium bicarbonate and 10 mMHEPES (Eagle-NaHCO3). No supplementary serumwas added to the media.

Vaccines and mitogens. For the preparation of avaccine of ultraviolet (UV)-killed bacteria an over-night culture of the bacterial strain in Standard-Inutrient broth was diluted (1:10) in fresh medium,and growth was permitted to the end of the logarith-mic growth phase. The bacteria were then centri-fuged for 20 min at 3,000 x g and resuspended in0.9% NaCl solution (saline).A layer (2 mm) of a suspension containing about 2

x 108 bacteria/ml in plastic petri dishes was irradi-ated with short-wave UV light for 20 min. The sus-pension was then centrifuged for 20 min at 2,000 x gand washed three times with cold saline. When usedfor stimulation of spleen cell cultures, the UV-killedbacteria were prepared on the same day on whichthe experiment was performed. Complete killing ofthe bacteria was checked by plating a sample ontoagar and with enrichment cultures in Standard-Inutrient.LPS was obtained from a single batch ofS. typhi-

murium F1826 by the phenol-water method (42).PHA (Difco Laboratories, Detroit, Mich.) was usedas a T-cell mitogen (26).

Determination of susceptibility to infection. S.typhimurium F1826 was grown in Standard-I nutri-ent broth to the end of the logarithmic growthphase. Seven bacterial suspensions, ranging from102 to 108 viable cells/ml, were prepared by dilutionin 10-fold steps with cold saline. Portions (0.1 ml) ofthese suspensions were injected intraperitoneallyinto mice (groups of eight each). The animals wereobserved up to day 30 after challenge. The meanlethal dose (LD5,,) values were determined by themethod of Reed and Muench (31).

Spleen cell cultures. The mice were killed withether anesthesia. The spleens were immediately re-moved aseptically and homogenized in a loosely fit-ting ground-glass homogenizer in cold Eagle-HEPES. The cells were collected by centrifugationat 180 x g for 7 min and washed twice in cold Eagle-HEPES. The dead cell count, as determined by thetrypan blue exclusion method, was normally lessthan 10%. The spleen cell suspension was adjustedto 107 nucleated cells/ml of Eagle-NaHCO3, and 1 x106 cells (0.1 ml) were pipetted into each well of apolystyrene microtiter tissue culture plate (C. A.Greiner und S6hne, Kunststoffwerke, 7440 Nurtin-gen, West Germany). Parallel cultures were set upwith spleen cells obtained from single mice; thespleens were only pooled when it was required bythe size of the experiments. To each well the appro-priate amount (see below) of mitogen was added in0.1 ml of Eagle-NaHCO3. The cultures were incu-bated in a CO2 incubator (W. C. Heraeus GmbH,6540 Hanau, West Germany) in a humidified mix-ture of 95% air and 5% CO2. After 48 h, 1 ,uCi of[methyl-3H]thymidine (specific activity, 18 Ci/mmol)(Radiochemical Centre, Amersham, England) in 50,ul of Eagle-NaHCO3 was added per well, and theincubation was continued for a further 16 h.

The cells were harvested automatically on glass-fiber filters (Reeve-Angel, grade 934 AH; Microbio-logical Associates, Inc., Bethesda, Md.) in a multipleautomatic sample harvester (MASH II apparatus;Microbiological Associates, Inc., Bethesda, Md.)by washing with deionized water. The filterswere air-dried, transferred to Packard glass vials,and counted in 10 ml of scintillation fluid usinga Tri-Carb liquid scintillation spectrometer (Pack-ard Instrument Co., Inc., Downers Grove, III.).The scintillation fluid (1) was composed of: 3 litersof xylol, 1 liter of Triton X-114, 12 g of PPO (2,5-diphenyloxazole) (compoundslobtained from C. Roth,75 Karlsruhe, West Germany), and 200 ml of de-ionized water.

RESULTSScreening of different strains of mice. In

preliminary experiments nine inbred strains ofmice and one F1 hybrid were screened for theirsusceptibility to intraperitoneal infection withS. typhimurium F1826 and the proliferativeresponse of their spleen cells to optimalamounts of LPS and PHA (Fig. 1). Spleen cellsof strains C3Hf, CBA, and (C57B1/6 x C3H)F1hybrids, which were the most resistant to theinfection, showed the highest incorporation of[3H]thymidine when stimulated with optimalamounts of LPS; spleen cells of the C3H/HeJmice, which were very susceptible to the infec-tion, showed the lowest stimulation with LPS.This result suggested a possible relationshipbetween the capacity of spleen cells of thesemouse strains to respond to the mitogenic effectof LPS and the resistance to infection with S.typhimurium.

For further studies strains C5B1/6, C3Hf, andC3H/HeJ were selected. Figure 2 shows theproliferative response of their spleen cells toincreasing amount of LPS. The incorporation oflabeled thymidine in unstimulated spleen cellswas different for the three strains of mice. Theextent of this spontaneous incorporation corre-lated neither with the maximum stimulation inthe presence of mitogen nor with the suscepti-bility of the animals to infection with S. typhi-murium.The sensitivity of the three mouse strains to

endotoxin and whole UV-killed bacteria, aswell as their susceptibility to the infection withS. typhimurium variants of different virulence,is compared in Table 1. The two strains, C57B1/6 and C3H/HeJ, both showing an LD50 of about10 S. typhimurium F1826 when injected intra-peritoneally, could be differentiated by the ef-fect of an intraperitoneal injection of S. typhi-murium F1826a or a subcutaneous injection ofS. typhimurium F1826. In both cases the C3H/HeJ mice were more susceptible to infectionthan the C57B1/6 mice, whereas C3Hf was themost resistant strain.

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28 VON JENEY, GUNTHER, AND JANN

strain C57B1/6 BIO.BR 81O.D2 C3Hf C3H/DiSn C3H.Sw C3H/H*J CBA/H OBAj

H-2 b k d k k b k k d

LDSO -10 -10 -10 2.0x104 1.2x10 8100 (10 1.2x -102.20l2FIG. 1. In vitro stimulation ofspleen cells of various inbred strains of mice with LPS (open bars) (optimal

amounts, 50 to 100 jg of LPS per ml of culture medium) and PHA (hatched bars) (10 pglml) andnonstimulated controls (crossed bars). Three to nine mice were used per strain. The LD5,, values of theintraperitoneal infection with S. typhimurium F1826 are shown.

40-

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C25-C I

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0 10I 10-2 10-1 ;10 10I 10 103 1Llpopolysaccharide (ug/lit) PHA (,uglrrd

FIG. 2. Dose response curves of the spleen cell stimulation ofC57B116, C3Hfand C3H/HeJ mice with LPS.Results are the average ofdata obtained with four mice for each strain. PHA stimulation (10 pglml) was usedas a control. Symbols: 0, C57B116; A, C3Hf; O, C3HIHeJ.

There seemed to be no direct correlation be-tween the sensitivity of the mice to the toxiceffect of endotoxin and their susceptibility toinfection with S. typhimurium. The C3H/HeJmice are the least sensitive to the toxicity ofLPS, the C3Hf mice take an intermediate posi-tion, and C57B1/6 mice are the most sensitive.Thymidine incorporation by spleen cells of

mice injected with LPS. During an infectionwith gram-negative bacteria and especially un-der the condition of septicemia, the animals areexposed to LPS (44). It was thus of interest toinvestigate the mitotic responsiveness of thespleen cells of LPS-treated mice. For this pur-pose mice of the three strains were injectedintravenously with 10 ,ug of LPS ofS. typhimu-

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rium F1826 at 18, 12, 9, 6 and 3 h, respectively,before the in vitro test. The results are shownin Fig. 3. The in vitro responsiveness to LPSand PHA of the spleen cells of C57BI/6 micethat had received LPS was reduced for about 18h. The minimum responsiveness occurredabout 6 h after the injection. This was not seenwhen spleen cells of the other two mousestrains were used. Essentially the same resultswere obtained after an intraperitoneal injectionof 10 ug of LPS.To test the dose dependency of the effect of

LPS pretreatment, mice of the three strainswere injected intravenously with variousamounts ofLPS 6 h before testing their prolifer-ative response in vitro. Increasing amounts ofintravenously administered LPS led to a de-crease in the response to LPS in all threestrains (Fig. 4), with reduction being most pro-nounced in C57BI/6 mice.With PHA as mitogen, suppression of thymi-

dine uptake was observed in the C57BI/6 andC3H/HeJ mice, whereas no such effect was seenin the C3Hf strain.

Since in the absence of mitogen the spleencells of mice injected with LPS did not incorpo-rate significantly more [3H]thymidine than didspleen cells of normal mice (Fig. 4), a carry-over of LPS could not be the reason for thealtered response to LPS in vitro. To rule out aneffect of LPS carry-over on the PHA response,LPS and PHA were added simultaneously tonormal C57BI/6 spleen cell cultures. Nosuppression, but rather a slight positive syner-gistic effect on the [3Hlthymidine incorpora-tion, was found (data not shown).The changes in the responsiveness to mito-

gens after an injection of 10 ug of LPS wereonly observed during the first 24 h. No effect onthe spleen cells was seen during a further ob-servation period of 15 days, measured on days3, 6, 9, 12, and 15.The above-described LPS effects are less pro-

nounced after an intraperitoneal injection ofLPS (data not shown).Thymidine incorporation by spleen cells of

mice immunized with IUV-killed bacteria. Tostudy the response of spleen cells to the intact

TABLE 1. Sensitivity ofmice to LPS and killed bacteria and susceptibility to infection with S. typhimurium

S. typhimurium F1826a killed Live S. typhimuriumbMouse strain LP& by LJW F1826 (i.p.) F1826 (s.c.) F1826a (i.p.)

C57BI/6 150 1.5 x 109 10 1 x 104 1 x 105C3Hf 600 2.0 x 109 2 x 104 3 x 106 6 x 105C3H/HeJ 1,500 7.5 x 109 <10 102 102

a LD50 amount (micrograms) of LPS injected intraperitoneally.b LD50 number of bacterial cells injected intraperitoneally (i.p.) or subcutaneously (s.c.) causing death of

mice.

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1PIG. 3. Change in the spleen cell stimulation in C57B116 (-), C3Hf (A), and C3HIHeJ (-) mice after anintravenous injection of10 pg ofLPS. Solid lines, LPS stimulation (50 pg/ml of culture medium); brokenlines, PHA stimulation (10 pg/ml ofculture medium); dotted lines, nonstimulated controls. The results arethe average of data obtained with two mice per experimental point.

0

Ef 30u0aE

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30 VON JENEY, GUNTHER, AND JANN

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FIG. 4. Dose-dependent changes ofthe spleen cell stimulation in C57B116 (a), C3Hf(A), and C3H/HeJ (A)mice 6 h after an intravenous injection of10 ug ofLPS. Solid lines, LPS stimulation (50 mg/ml); broken lines,PHA stimulation (10 pglml); dotted lines, nonstimulated controls. The results are the average of dataobtained with three mice per experimental point.

pathogen, UV-killed bacteria were used. UVirradiation renders the bacteria noninfectiveand nonproliferative, while preserving an un-denaturated surface. Under the assumptionthat whole bacteria, as their extracts, aremitogenic for mouse spleen cells, we testedthe in vitro response of the three strains ofmiceto a wide range (5 x 104 to 5 x 109 bacteria/ml ofculture medium) of whole UV-killed S. typhi-murium. It was found that these bacteria ex-erted only a slight mitogenic effect on thespleen cells, with a maximum stimulation ratioof 1.3 (manuscript in preparation). This lowresponse was not due to a detrimental effect ofthe bacteria on the spleen cells in culture, sincea normal response to LPS and PHA was ob-tained when they were added to the culturestogether with the UV-killed bacteria.

In another experiment viable S. typhimu-rium bacteria were added to the spleen cellcultures. The bacteria were rapidly killed bythe antibiotics in the medium, as determinedby dilution in saline and plating on agar. Thespleen cell cultures remained clear and the pHdid not drop. A low incorporation of labeledthymidine, of the same order as with UV-killedbacteria, was obtained with 2 x 106 to 2 x 107viable bacteria/ml. This shows that the lowresponse to the UV-killed bacteria probablywas not due to UV irradiation of the bacteria.UV irradiation of LPS did not influence itsmitogenicity.The intraperitoneal injection of 1 x 108 UV-

killed S. typhimurium F1826 into C57BI/6,

C3Hf, and C3H/HeJ mice rendered them specif-ically immune (survival at 30 days) to an intra-peritoneal challenge with 100 LD5,, of S. typhi-murium F1826 on days 5, 10, 15, and 30 afterimmunization (unpublished data). Thus, onseveral days after immunization with UV-killed bacteria, spleen cells of these mice werestimulated in vitro with LPS, UV-killed bacte-ria, and PHA. No effect of the immunization onthe in vitro response to LPS and PHA wasfound in the three strains of mice. However, asignificant proliferative response was obtainedwith UV-killed bacteria as stimulant on day 4,which remained elevated up to day 28 (Fig. 5).We wanted to know whether this effect was

specific for the 0 antigens, which are promi-nent antigens on the surface of gram-negativebacteria. To test this, we used UV-killedE. coli0111, which is unrelated to S. typhimuriumwith respect to the 0 antigens, as stimulant invitro. These bacteria, when added to culturesof the spleen cells of C57BI/6 mice that hadbeen injected with UV-killed S. typhimuriumF1826a, gave rise to a proliferative responsecomparable to that obtained with the homolog-ous bacteria (Fig. 5). Therefore, the enhancedresponse to UV-killed bacteria is not specificfor the 0 antigens of the bacteria and may bedue to other bacterial surface structures.

DISCUSSIONUpon screening nine inbred strains of mice

and one F1 hybrid, we found that the strainsmost resistant to intraperitoneal infection with

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STIMULATION OF MOUSE SPLEEN CELLS

S. typhimurium, viz., C3Hf, CBAIH, and the(C57B1/6 x C3Hf) F1 hybrid, also showed thehighest proliferative response of their spleencells to LPS in vitro. In C57B1/6, C3Hf, andC3H/HeJ strains, which were examined moreclosely, the same association could be observedwhen the mice were infected by the subcuta-neous route. These results indicate that theremay exist a correlation between the capacity ofthe spleen cells of mice to respond to the mito-genic action of LPS and the resistance to infec-tion with Salmonella. This would add anotherparameter to those described by previous au-thors, when studying differences in the suscep-tibility to infection. Thus, inbred strains ofmice have been found to differ in their reticulo-endothelial system function and differentialleukocyte count (5), in the number or activity oftheir macrophages (12, 23), or in their responseto immunization with Salmonella antigens,measured either as protection from infection(32) or as delayed hypersensitivity reaction(30).The C3H/HeJ mice are of particular interest

in our studies. In spite of their insensitivity tothe toxic effect ofLPS (38), C3H/HeJ mice werefound to be highly susceptible to infection withS. typhimurium. This is in keeping with theresults of Robson and Vas (32) and shows thatthe toxic effect of LPS may be differentiated

from the detrimental effect that the viable Sal-monella exert on their host.

UV-killed bacteria and bacteria killed by an-tibiotics, i. e., bacteria with intact cell surfaces,hardly induced the stimulation of spleen cells.Since the cell walls of gram-negative bacteriaare known to contain compounds that are mito-genic for B-cells, such as lipid A (2, 34) and alipoprotein (25), these must be cryptic in theintact bacteria.

All three strains of mice showed a transientdose-dependent decrease of their spleen cell re-sponse to LPS after an intravenous injection ofincreasing amounts of LPS, whereas PHA re-sponsiveness was only decreased in the infec-tion-susceptible C57B1/6 and C3H/HeJ mice.The results can be explained either by a loss ofcells normally responsive to these mitogensfrom the spleen or by inactivation ofthese cells.It is known that LPS may suppress the immuneresponse (19, 35), and a depletion of T-cell-de-pendent areas in the spleen after LPS injectionhas been reported (23). Since LPS may be re-leased into the bloodstream during localized orgeneralized gram-negative infection (44) or en-ter from the intestinal tract (12), these effects ofLPS on spleen cells may be ofimportance to theoutcome of the infection.The response to UV-killed bacteria in vitro,

which in nonpretreated mice was hardly meas-

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FIG. 5. Spleen cell stimulation in C57B116, C3Hf, and C3HIHeJ mice after immunization with UV-killedS. typhimurium F1826a (1 x 108 bacteria/mouse, intraperitoneally). The results are the average of dataobtained with three mice per experimental point. The open symbols and the hatched bars are the stimulation ofnonimmunized controls. (a, b, and c) Stimulation with UV-killed S. typhimurium F1826a; (d) stimulationwith UV-killed E. coli 0111. The LPS stimulation (50 pg/ml of culture medium) and PHA stimulation (10pg/ml) of nonimmunized control mice are indicated with open symbols.

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32 VON JENEY, GUNTHER, AND JANN

urable, was enhanced markedly by immuniza-tion in all three strains of mice. This increasewent through a maximum on days 4 to 7 andstill was measurable on day 28. The in vitroresponse to LPS and PHA, however, remainedvirtually unchanged during this time. An en-hanced response was also obtained with E. coli0111 as stimulant, having an 0 antigen that iscompletely different from that of S. typhimu-rium. If one argues that the enhanced responseis due to specific recognition by memory cells,the structures recognized must be differentfrom the 0 antigenic polysaccharides. In an-amnestic in vitro responses, T-lymphocytesplay a decisive role, and preliminary results in-deed indicate that this may be the case in ourexperiments, since pretreatment with anti-Thy-1.2 serum and complement reduced the en-hanced response to UV-killed bacteria (manu-script in preparation). This would be in keepingwith earlier reports in which the participationof T-cells in the defense against intracellularinfections was demonstrated (7, 27, 28). Ourresults with UV-killed bacteria may serve as abasis in the search for bacterial surface compo-nents that are relevant in infection and that arerecognized by the immune system ofthe host.

LITERATURE CITED

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