Vol. 56, No. 6INFECTION AND IMMUNITY, June 1988, p. 1436-14410019-9567/88/061436-06$02.00/0Copyright © 1988, American Society for Microbiology

Natural Killer Cell Activation and Interferon Production byPeripheral Blood Lymphocytes after Exposure to BacteriaGARY R. KLIMPEL,* DAVID W. NIESEL, MARIA ASUNCION, AND KATHLEEN D. KLIMPEL

Department of Microbiology, University of Texas Medical Branch, Galveston, Texas 77550

Received 7 January 1988/Accepted 19 February 1988

We have previously shown that peripheral blood natural killer (NK) cells have significant levels of cytotoxicactivity against Shigellaflexneri-infected HeLa cells. In this report, we show that NK cell activity against K562tumor cells and Shigellaflexneri-infected HeLa cells can be greatly enhanced by preincubating peripheral bloodlymphocytes (PBL) for 18 h with kanamycin-treated Shigella flexneri or Salmonella typhimurium. Cell-freesupernatants obtained from PBL-bacteria cultures contained high levels of interferon (IFN) activity, which wascharacterized as a mixture of IFN--y and IFN-a. Cytotoxic activity associated with PBL precultured withshigellae was associated with predominantly CD16+ (Leu-ll+) and CD2+ (OKT-ll+) cells. Further, IFNproduction was dependent upon the presence of CD16+ and CD2+ cells at culture initiation. Enhancement ofcytotoxic activity associated with PBL-bacteria cultures did not, however, appear to be dependent upon IFNproduction, since low numbers of bacteria which failed to stimulate IFN production induced high levels ofNKcell activity. Lipopolysaccharide appeared not to be involved in bacteria-induced IFN production or enhancedNK cell activity, since Salmonella lipopolysaccharide failed to induce IFN production or enhance NK cellactivity. These results suggest that IFN production by NK cells and the killing of bacteria-infected cells play animportant role in host defense against facultative intracellular bacterial infections.

The importance and exact role of natural killer (NK) cellsand interferon (IFN) in host defense against facultativeintracellular bacterial infections is currently unclear. A num-ber of studies have reported that lymphocytes possessing aphenotype closely resembling NK cells might be directlyinvolved in the direct killing of bacteria (14, 16-18, 24).Additionally, lymphokine (IFN, etc.) production by NKcells or T cells may play an important role in enhancing thebactericidal activity of monocytes and neutrophils and mayalso contribute to other host defenses against differentbacterial infections (6, 15, 19). We (19) and others (2, 3) haveshown that the pretreatment of human epithelial cells ormouse fibroblasts with natural and recombinant IFNs resultsin a cellular state resistant to subsequent invasion by salmo-nellae or shigellae. Further, NK activity is increased in boththe spleen and lung early in Chiamydia trachomatis infectionof both resistant and susceptible mice (31). Recently, non-major histocompatibility complex (non-MHC)-restricted cy-totoxic activity of NK cells was greatly enhanced by thecontact of lymphocytes with glutaraldehyde-fixed Salmo-nella spp. (25, 26). IFN production was also shown to beassociated with this enhanced NK cell activity (25). In thisregard, we have recently shown that lymphocytes whichappear to be NK cells preferentially kill Shigella flexneri-infected HeLa cells (11). This cytotoxic activity was greatlyenhanced by pretreatment of peripheral blood lymphocytes(PBL) with either IFN-ot or interleukin-2 (IL-2). These datasuggest that the killing of bacterium-infected cells by NKcells plays an important role in host defense against infec-tions mediated by facultative intracellular bacteria. Alterna-tively, since the destruction of bacteria-infected cells mayproduce significant tissue damage, NK cells may signifi-cantly contribute to the pathogenesis associated with suchinfections.

In this report, we show that non-MHC-restricted cyto-toxic activity against K562 tumor cells and S. flexneri-

* Corresponding author.

infected HeLa cells is greatly enhanced by preincubation ofPBL with either shigellae or salmonellae. This cytotoxicactivity was mediated by CD2+ and CD16+ cells. PBLcultured with high numbers of bacteria also produced highlevels of IFN. The significance of this cytotoxic activity, itsenhancement by contact with bacteria, and bacterial induc-tion of IFN production are discussed with regard to hostdefense against facultative intracellular bacterial infections.

MATERIALS AND METHODSReagents and tumor cell lines. Partially purified human

IL-2 was purchased from Electro-Nucleonics, Inc., Colum-bia, Md. This preparation had 600 U of IL-2 activity per mland no detectable IFN activity. Partially purified humanIFN-a was obtained from Lee Biomolecular Research, Inc.,San Diego, Calif. IFN-ot contained 2.3 x 105 U of antiviralactivity per ml (2.1 x 105 U/mg). Anti-Leu-11 was obtainedfrom the Becton Dickinson Monoclonal Center, Inc., Moun-tain View, Calif. Anti-OKT-11 and anti-OKT-3 were ob-tained from Ortho Pharmaceutical Co., Rantan, N.J. Anti-sera against human IFN-ot (G026-502-568), human IFN-r(G028-501-568; 3,000 neutralizing U/ml), and human IFN--y(G034-501-565; 1,300 neutralizing U/ml) were obtained fromthe National Institutes of Health (NIH), Bethesda, Md.Phenol-extracted and chromatographically purified lipopoly-saccharide (LPS) from Salmonella typhimurium and Esche-richia coli was obtained from Sigma Chemical Co., St.Louis, Mo. Rabbit low-tox complement for human lympho-cytes was purchased from Cedarlane Laboratories Limited,Hornby, Canada. HeLa cells were obtained from the Amer-ican Type Culture Collection, Rockville, Md. HeLa cellswere maintained as suspension (spinner) cultures withSMEM (GIBCO Laboratories, Grand Island, N.Y.) supple-mented with 7% fetal calf serum (FCS) and antibiotics. K562tumor cells were maintained as suspension cultures withRPMI 1640 medium plus 10% FCS and antibiotics.

Bacterial infection of HeLa cells. Invasive S. flexneri (se-rotype 2A) and S. typhimurium were maintained at -70°C,

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and new cultures were prepared weekly. Shigella sp. strainSA100 efficiently invades HeLa cells and kills 2-day-oldchicken embryos within 24 h (50% lethal dose, 5 CFU) (11).

Shigella sp. strain SA100 or a Salmonella strain was splitfrom overnight cultures into brain heart infusion broth andharvested at the mid-logarithmic phase of the culture. Bac-teria were then washed with RPMI 1640 medium with 2 ,ug ofFeCl3 per ml and 0.45% glucose (RPMI-Fe) and then sus-pended as 20x stocks in RPMI-Fe. Before invasion oraddition to PBL cultures, bacteria were diluted to 108 to 2 x1010 CFU/ml in RPMI-Fe. Bacterial cells were enumeratedby dilution and plating on Luria agar (L agar). In someexperiments, bacteria were heat killed. Bacteria were dilutedin supplemented RPMI 1640 medium (20 ,ug of streptomycin,20 U of penicillin, and 50 ,ug kanomycin per ml and 10%FCS). Suspensions were brought rapidly to 70°C, maintainedfor 20 min, and then cooled to room temperature before use.

Suspension cultures of HeLa cells were maintained at 4 x105 cells per ml. For bacterial invasion, equal volumes ofHeLa cells (0.5 ml, 2 x 106 HeLa cells) and shigellae(SA100, 2 x 109 CFU) or HeLa cells and medium weremixed together in centrifuge tubes (13 by 100 mm). Thesemixtures were then incubated at 37°C for 90 min withconstant agitation. HeLa cells were then washed six timeswith RPMI 1640 medium containing antibiotics and kanamy-cin (50 ,ug/ml). HeLa cells were then labeled with 51Cr for 1h in RPMI 1640 medium containing antibiotics and kanamy-cin. Different 51Cr-labeled cell populations were then as-sessed for the presence of intracellular bacteria and for theirsusceptibility to PBL-mediated cytotoxic activity by a 51 Cr-release assay as previously described (11).More than 80% of the HeLa cells incubated with invasive

shigellae, as described above, contained one or more shigel-lae. This was determined by plating 500 HeLa cells anddetermining the number of infected cells with an agarose-agar overlaying procedure as previously described (19).

In vitro culture of PBL. Human peripheral blood wasobtained from randomly chosen normal donors, and mono-nuclear cells were isolated with Ficoll-Paque (PharmaciaFine Chemicals, Piscataway, N.J.) density gradient centrif-ugation. Donors had no known prior history of shigellosis.PBL were cultured in RPMI 1640 medium containing 10%FCS and kanamycin (50 ,ug/ml). Two million PBL in 0.5 mlof medium were incubated for 18 h with one of the following:(i) shigellae, (ii) salmonellae, (iii) various concentrations ofSalmonella LPS or E. coli LPS, (iv) different volumes ofculture supernatants, or (v) IL-2 (200 U/ml). Under theabove culture conditions, shigellae and salmonellae wererendered nonviable in <4 h. At the end of culture, cells andcell-free supernatants were obtained from pooled duplicateor triplicate cultures. Cells were washed twice in RPMI 1640medium before being assessed for NK cell activity againstdifferent target cells. Cell-free supernatant was stored at-70°C until being assayed for the presence of IFN or IL-2activity.IFN assay. IFN activity was measured on human WISH

cells by a cytopathic effect inhibition assay with Sindbisvirus (19). One unit of IFN is defined as the amount thatreduces the number of plaques or cytopathic effect by 50%compared with that in the virus control. IFN titers weredetermined relative to an NIH reference standard and wereexpressed as NIH reference units per milliliter.To characterize the IFN present in different supernatants,

IFN-containing supernatants were incubated with eithermedium or anti-IFN antisera for 1 h at 25°C. Antisera wereused at concentrations sufficient to completely neutralize the

IFN activity. Treated samples were then assayed for IFNactivity as previously described.

Cytotoxic assay. The cytotoxic activity of different PBLcell populations against HeLa cells and K562 tumor cellswas assessed by a 4-h microcytotoxicity assay with 51Cr-labeled target cells. This assay and the procedure for labelingtarget cells have been fully described (11). In brief, 104 to 106effector cells were mixed with 104 51Cr-labeled target cells inconical-well microdilution plates in a reaction volume of 0.2ml of medium containing antibiotics and kanamycin. Aftercentrifugation (30 s, 400 x g), the cultures were incubated at37°C for 4 h. A sample of cell-free supernatant was thentaken, and its 51Cr content was determined in an automaticgamma counter. The cytotoxic activity was determined asfollows: cytotoxic activity = [(experimental 51Cr release -

spontaneous 51Cr release)/(total cell-associated 51Cr)] x 100.Data are expressed as either the percentage of 51Cr releaseor the lytic units per 107 PBL and were calculated aspreviously described (22). A lytic unit is defined as thenumber of cells required to cause 25% cytotoxic activity.Antibody and complement treatment of PBL. Leu-11+

(CD16), OKT-11+ (CD2), and OKT-3+ (CD3) cells wereeliminated from PBL cell populations by two successivetreatments with antisera and rabbit complement. PBL orPBL pretreated with shigellae were incubated for 45 min at4°C with each antiserum (2 jig/ml). This cell suspension (5 x106 cells per ml) was washed once with RPMI 1640 mediumand was incubated for 45 min at 37°C with a 1/10 final dilutionof rabbit complement. PBL were then washed once withRPMI 1640 medium and were incubated for an additional 45min at 37°C with each antiserum and rabbit complement.PBL were then washed twice with RPMI 1640 medium plusFCS and were either used as effector cells in the 51Cr-releaseassay or assessed for their ability to produce IFN or generateenhanced NK cell activity.

RESULTS

Shigellae and salmonellae induced IFN production andenhanced NK activity. The ability of bacteria to enhance NKcell activity against different target cells was investigated byincubating PBL with various numbers of shigellae or salmo-nellae for 18 h in the presence of kanamycin. Cells obtainedfrom these cultures were assessed for NK activity against51Cr-labeled target cells (HeLa cells, S. flexneri-infectedHeLa cells, or K562 tumor cells). Cell-free supernatantsfrom these cultures were also obtained and assessed for IFNand IL-2 activity. Results from different representative ex-periments are shown in Fig. 1 and 2 and in Table 1. PBLincubated 18 h with either shigellae or salmonellae hadenhanced cytotoxic activity against all three target cells.HeLa cells infected with S. flexneri, however, were muchmore susceptible to killing compared with untreated HeLacells (Table 1). Cell-free supernatants obtained from culturescontaining PBL and high numbers (.106) of shigellae orsalmonellae contained significant levels of IFN activity.Although these supernatants routinely contained high levelsof IFN activity, no IL-2 activity could be detected in thesesupernatants (data not shown). IFN production, however,did not appear to be necessary for bacteria-induced NK cellactivation, since cultures containing low numbers of bacteriaresulted in NK cell activation without detectable IFN pro-duction (Fig. 1 and 2). We attempted to directly test thispossibility by adding antibody to IFN-ao or IFN-y to PBL-bacteria cultures and assessing IFN and NK cell activity at18 h. Polyclonal (goat and rabbit) antisera and control sera

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Number of Salmonella per culture

FIG. 1. NK cell activation and IFN production by PBL culturedwith S. typhimurium. PBL (2 x 106) were cultured with variousnumbers of salmonellae in medium supplemented with antibiotics.After 18 to 24 h of culture, supernatants were collected and assessedfor IFN activity (K) and PBL from each culture were assessed forNK cell activity against K562 tumor cells. NK cell activity isexpressed in lytic units per 107 PBL (L).

obtained from NIH were tested. Results from these experi-ments were uninterpretable, since the addition of equivalentconcentrations of NIH control sera (eight different experi-ments) always inhibited not only NK cell activation but alsoNK cell activity in control cultures (PBL plus medium).PBL incubated 18 h in supernatants, which contained IFN

and were obtained from PBL-bacteria cultures, had en-

hanced NK cell activity against S. flexneri-infected HeLacells (Table 1). This enhancement appeared to correlate withthe amount of IFN activity present in the supernatant.Supernatants from control cultures, from cultures containingbacterial numbers (.2.5 x 105) too low to induce IFNproduction (but not too low to enhance NK cell activity),and from PBL-bacteria cultures which were tested at dilu-tions at which the IFN activity was <50 U/ml failed toenhance NK cell activity (data not shown).

500

700

400

I400

:300-

- z

C 200

2. ~~~~~~~~~~~~~~~~~40

100-~~~~~~~~~~~~~~~~1T<10

Medium Sx104 10 2.5x105 5x10 10" 2.5x10 5x10 107only

Number of Shigella per culture

FIG. 2. NK cell activation and IFN production by PBL culturedwith S. flexneri. PBL (2 x 106) were cultured with various numbersof shigellae in medium supplemented with antibiotics. After 18 to 24h of culture, supernatants were collected and assessed for IFNactivity (K) and PBL from each culture were assessed for NK cellactivity against K562 tumor cells. NK cell activity is expressed inlytic units per 107 PBL (OI).

TABLE 1. NK cell activity enhancement byPBL-bacterium cultures

% 51Cr release IFNEffector/ (target cell assessed) (U/ml)PBL culture target in

incubation" ratio HeaHeLa + K52culturesHeLa S.flexneri K562 at 18 h

Expt 1Medium alone 50:1 8 19 42 <10

12:1 2 8 18

S. flexneri 50:1 16 49 65 8512:1 3 25 29

S. typhimurium 50:1 12 35 62 3012:1 2 14 34

S. typhimurium LPS 50:1 46 <10(100 pg/ml) 12:1 16

S. typhimurium LPS 50:1 45 <10(10 ,ug/ml) 12:1 12

Heat-killed S. flexneri 50:1 70 4812:1 38

Expt 2Medium alone 50:1 5 20 60 <10

12:1 3 13 36

Supernatant from 50:1 14 49 78 800PBL + S.flexneri 12:1 8 27 49

Supernatant from 50:1 6 29 68 <10PBL + medium 25:1 4 22 51

12:1 4 17 42

'PBL (2 x 106) were cultured in medium (0.5 ml) containing 50 jig ofkanamycin per ml for 18 h with (i) nothing added, (ii) 5 x 106 S. flexneri, (iii)5 x 106 heat-killed S.flexneri, (iv) 5 x 106 S. typhimurium, (v) S. typhimuriumLPS (10 or 100 jig/ml), (vi) supernatant (50% [vol/vol]) from an 18-h culturecontaining PBL and S. flexneri and having 2,000 U of IFN activity per ml, or(vii) supernatant (50% [vol/vol]) from 18-h cultures containing PBL andmedium and having <10 U of IFN activity per ml.

Heat-killed bacteria also induced IFN production and NKcell activation (Table 1). LPS, however, appeared not to beinvolved in either salmonella- or shigella-induced NK cellactivation or IFN production. PBL cultured 18 h withSalmonella LPS (1 to 100 ,ug/ml) had no enhanced NK cellactivity or IFN production (Table 1). These results wereobserved in experiments with PBL from eight individuals. Inthis regard, we have also previously reported that LPS-containing culture supernatants from Shigella cultures alsofailed to induce NK cell activation (11).We have recently also investigated whether E. coli LPS

could enhance NK cell activation or induce IFN productionin PBL. Very inconsistent results were obtained from theseexperiments. PBL from 18 individuals were investigated forNK cell activation following in vitro culture with variousconcentrations of E. coli LPS. PBL from 11 of these indi-viduals showed no enhancement of NK cell activity. PBLfrom seven of these individuals, however, did display en-hanced NK cell activity following in vitro culture with E. coliLPS (data not presented).The results presented in Table 1 and in Fig. 1 and 2 suggest

that contact between PBL and either shigellae or salmonel-lae can lead to enhanced NK cell activity against differenttarget cells, including S. flexneri-infected HeLa cells. Whenhigh numbers of bacteria are present, PBL produce signifi-

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TABLE 2. Characterization of the IFN produced byPBL cocultured with S. flexneri

IFN (U/ml) in supernatant pretreated with:Source of Anti-

Expt IFN-containing Anti- Anti- Anti- IFN-a +supernatanta Nothing IFN-a IFN-p IFN-y Anti-

IFN-y

1 PBL + IL-2 20 18 22 <5PBL + PHA 500 500 480 <5

2 PBL + S.flexneri 300 60 280 20 <103 PBL + S.flexneri 50 10 30 <10 <10a PBL (2 x 106) were cultured for 18 h with IL-2 (200 U/ml), 5 x 106 S.

flexneri, or phytohemagglutinin (PHA) (10 ,ug/ml). Cell-free supernatant fluidsfrom these cultures were then assessed for IFN activity, and the type of IFNpresent in each supernatant was characterized with the appropriate antiserumas described in Materials and Methods.

cant levels of IFN activity, which also may play an impor-tant role in stimulating NK cell activity.

Shigella-induced IFN-a and IFN--y production in PBL. Thetype of IFN produced by PBL incubated for 18 h withshigellae was determined with type-specific antisera toIFN--y, IFN-,B, and IFN-a. The IFN produced by shigella-stimulated PBL was a mixture of IFN-a and IFN--y (Table 2).In contrast, PBL incubated with IL-2 produced low levels ofIFN--y, which was neutralized by antiserum to IFN--y.

Characterization of cytotoxic cells present in cultures ofPBL plus shigellae. The phenotype of the cytotoxic effectorcells present in cultures containing PBL plus shigellae wasinvestigated by antibody and complement lysis with OKT-11, OKT-3, and Leu-11 antisera. Fresh PBL and PBLcocultured for 18 h with shigellae were treated with twocycles of antibody and complement, and the resultant cellswere assessed for cytotoxic activity against K562, HeLa,and S. flexneri-infected HeLa cells. Treatment of both cellpopulations with either anti-OKT-11 and complement oranti-Leu-11 and complement almost totally eliminated(.80%) cytotoxic activity against all target cells (Table 3).

TABLE 3. Characterization of cytotoxic cells present incultures of PBL plus S. flexneri

% 51Cr release by target cell':

Effector cells Treatment of S6 flexneri-effector cells' K562 infected HeLaHeLa

Fresh PBL Untreated 35 16 4C 36 15 4Anti-CD16 + C 5 (86) 3 (80) 0Anti-CD2 + C 0 (94) 1 (94) 0Anti-CD3 + C 41 (0) 20 (0) 8

PBL + S. flexneri Untreated 51 50 20C 58 46 15Anti-CD16 + C 10 (83) 9 (80) 2Anti-CD2 + C 4 (94) 7 (85) 2Anti-CD3 + C 60 (0) 52 (0) 18

a PBL or PBL cocultured for 18 h with S. flexneri, as described in Table 1,were treated with anti-Leu-11, anti-OKT-3, or anti-OKT-11 serum pluscomplement (C) as described in Materials and Methods.

b Results are given for an effector/target cell ratio of 50:1 for both HeLacells and S. flexneri-infected HeLa cells and 12:1 for K562 tumor cells.Numbers in parentheses represent the percent reduction in cytotoxic activitycompared with complement control. Results presented are from one experi-ment which was representative of three different experiments.

TABLE 4. Characterization of cells necessary forshigella-induced IFN production

PBL IFN (U/ml) after pretreatment of PBL with':Expt culture Anti- Anti- Anti-

treatment Nothing C CD16 + C CD2 + C CD3 + C

1 Medium <10 <10 <10 <10S. flexneri 320 320 20 320IL-2 160 80 <10 640

2 Medium <10 <10 <10 <10 <10S. flexneri 200 240 <10 <10 153

3 Medium <10 <10 <10 <10 <10S. flexneri 267 130 <10 <10 234

a PBL were treated with different antisera plus complement (C) as de-scribed in Materials and Methods. The resultant cells were incubated with S.flexneri as described in Table 1. After 18 h of culture, cell-free supernatantswere collected and assessed for IFN activity.

These results strongly suggest that NK cells are the cyto-toxic effector cells present in cultures of PBL and shigellae.

Characterization of cells necessary for shigella-induced IFNproduction and NK cell activation. Cytotoxic activity incultures of PBL plus shigellae appeared to be mediated byNK cells (CD16+, CD2+, and CD3-). Experiments werethen conducted to determine whether NK cells were neces-sary for IFN production or were the precursor cells of theshigella-induced NK cell activity. Fresh P3L were treatedwith different antisera plus complement, and the resultantcells were cocultured with shigellae as previously described.After 18 h of culture, cell-free supernatant was obtained andassessed for IFN activity, and cells from these cultures wereassessed for NK cell activity against K562 tumor cells. PBLdepleted of CD16+ or CD2+ cells failed to produce any IFNactivity after 18 h of culture with shigellae (Table 4). Incontrast, T-cell-depleted PBL (CD3- cells) produced IFNlevels equivalent to the complement-treated controls. Inaddition, NK cell activity was absent in cultures of PBLdepleted by CD16' and CD2+ cells (Table 5). These resultssuggest that NK cells (CD16+ and CD2+) are the cellsproducing the IFN in cultures containing PBL and shigellaeand that they are the cells whose cytotoxic activity isenhanced.

DISCUSSIONWe have previously shown that lymphocytes which ap-

pear to be NK cells preferentially kill S. flexneri-infected

TABLE 5. Characterization of cells necessary forshigella-induced enhanced NK cell activity

% 5"Cr release afterpretreatment of PBL witha:........Effector/

Culture conditions Efetrtarget ratio Anti- Anti- Anti-

Nothing C CD16 CD2 CD3+C +C +C

PBL + medium 100:1 47 47 13 23 5125:1 16 23 5 10 28

PBL + S. flexneri 100:1 69 59 14 11 6225:1 48 38 4 2 54

aPBL were treated as described in Table 4. After 18 h, cells from eachculture were assessed for cytotoxic activity against K562 tumor cells. Resultspresented are from one experiment which was representative of three differentexperiments. C, complement.

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HeLa cells (11). This non-MHC-restricted cytotoxic activitywas dependent upon bacterial invasion of the HeLa cells andwas greatly enhanced by the pretreatment of PBL with IL-2or IFN-at. In the present study, we show that non-MHC-restricted cytotoxic activity against S. flexneri-infectedHeLa cells is also greatly enhanced by preculturing PBLwith either shigellae or salmonellae. This enhancement doesnot stem from cellular invasion of PBL, since these cells are

refractory to bacterial invasion (data not shown) and ka-namycin rapidly destroys the invasive activity of both ofthese bacteria. Bacteria-induced NK cell enhancement ap-peared to require the presence of bacterial cells, since LPSand culture supernatants from PBL-bacteria cultures whichcontained no IFN activity failed to enhance NK cell activityor induce IFN production. NK cell activation and inductionof IFN production by heat-killed shigellae, however, sug-gested that bacterial metabolism is not necessary. Thecomponent(s) of the bacteria which is involved in activatingNK celis and inducing IFN production is currently underinvestigation.The cells mediating bacteria-enhanced cytotoxic activity

appeared to be NK cells, since the cytotoxic activity againstboth K562 tumor cells and S. flexneri-infected HeLa cellswas almost completely eliminated after the removal of CD2+and CD16+ cells. The purification of these cells and a more

comprehensive phenotypic analysis are necessary for a more

complete identification of these cytotoxic effector cells andtheir precursors. In fact, the non-MHC-restricted killing ofdifferent tumor cell targets is mediated by a heterogeneouspopulation of cells (4, 8, 12, 13). However, the predominanteffectors of non-MHC-restricted cytotoxicity present in pe-ripheral blood appear to be NK cells which express CD16and Leu-19 (7, 12, 20, 21). Recently, Tarkkanen et al. (25)showed that after an 18-h culture with salmonellae, NK-cell-mediated, non-MHC-restricted killing of NK-sensitive and-insensitive targets was greatly enhanced. PBL and largegranular lymphocytes also produced high levels of IFN whenincubated for 18 h with glutaraldehyde-fixed salmonellae(26). In our studies, high levels of antiviral activity whichappeared to be a mixture of IFN-y and IFN-a was producedby PBL when the lymphocytes were incubated for 18 h withshigellae or salmonellae. No IL-2 activity was observed inthese culture supernatants (data not shown). Significantlevels of IL-2 activity, however, were detected in thesecultures at 48 h (data not shown). The enhanced non-MHC-restricted cytotoxic activity associated with PBL culturedfor 18 h did not appear to be dependent upon the productionof IFN. This conclusion is based on the fact that lownumbers of bacteria generally enhanced NK cell activitywithout inducing IFN production. We cannot, however, ruleout the possibility that undetectable levels of IFN--y orIFN-at act synergistically to enhance NK cell activity. In thisregard, Weigent et al. (29) have shown that mixtures ofIFN-at and IFN--y act synergistically in enhancing the NKcell activity of human PBL. Recently, Bandyopadhyay et al.(1) have shown that HLA-DR+ cells produce IFN-a whencocultured with cytomegalovirus-infected targets and thatthis IFN-a was necessary for stimulating the NK-cell killingof cytomegalovirus-infected targets.The cell type actually producing IFN-y or IFN-at after in

vitro culture with shigellae or salmonellae is not known. NKcells (CD2+ and CD16+) appear to be necessary for IFNproduction, but this requirement may reflect a necessaryinteraction between NK and other cells and/or solublefactors produced by NK cells which then induces IFNproduction in some other cell type. Additionally, it is not

known whether a single cell type can produce both IFN-,yand IFN-a concurrently. Recently Hughes and Baron (lOa)have shown that IFN-y can induce IFN-ot production indifferent lymphoid cells. Thus, bacteria may induce lowlevels of IFN--y, which in turn induces IFN-a production.The exact levels of these IFNs in the culture supernatantsare also difficult to determine, since the antiviral activity ofthese mixtures results from the synergistic interaction ofthese IFNs (5).

Bacteria-induced IFN production by PBL may also playother important roles in host defense against bacterial infec-tions. We (19) have shown that the pretreatment of HeLacells or mouse fibroblasts with IFN-ot or IFN--y results in acellular state resistant to subsequent invasion by salmonellaeor shigellae. Further, other studies have demonstrated thatIFN can inhibit a variety of intracellular pathogens both invitro and in vivo. The intracellular growth of protozoans (23,28), rickettsia (27), chlamydia (9), and legionella (30) spp. isrestricted by treatment with IFNs. We have also recentlyshown that murine and human primary cell lines producehigh levels of IFN-P as a consequence of Shigella infection(10). Therefore, IFN produced by PBL or bacteria-infectedfibroblasts could enhance the NK cell killing of bacteria-infected cells or protect adjacent cells from subsequentinfection by the bacteria.The ability of bacteria to induce IEN production and

enhance NK cell killing of bacteria-infected cells may playan important role in host defense against enteroinvasivebacterial infections. Alternatively, these host immune func-tions, if they do indeed occur in vivo, could contribute to thepathogenesis associated with such infections. Thus, NK cellactivity may contribute to the extensive localized tissuedestruction associated with enteroinvasive bacterial infec-tions.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants Al24677 and Al 23731 from the National Institutes of Health.

LITERATURE CITED1. Bandyopadhyay, S., B. Perussia, G. Trinchieri, D. S. Miller, and

S. E. Starr. 1986. Requirement for HLA-DR+ accessory cells innatural killing of cytomegalovirus-infected fibroblasts. J. Exp.Med. 164:180-195.

2. Bukholm, G., and M. Degre. 1983. Effect of human leukocyteinterferon on invasiveness of Salmonella species in HEp-2 cellcultures. Infect. Immun. 42:1198-1202.

3. Bukholm, G., and M. Degre. 1985. Effect of human gammainterferon on invasiveness of Salmonella typhimurium in HEp-2cell cultures. J. Interferon Res. 5:45-53.

4. Damle, N. K., L. V. Doyle, and E. C. Bradley. 1986. Interleukin2-activated human killer cells are derived from phenotypicallyheterogeneous precursors. J. Immunol. 137:2814-2822.

5. Fleischmann, W. R., Jr., J. A. Georgiades, L. C. Osborne, andH. M. Johnson. 1979. Potentiation of interferon activity bymixed preparations of fibroblast and immune interferon. Infect.Immun. 26:248-253.

6. Gomez, J., B. Pohajdak, S. O'Neill, J. Wilkins, and A. H.Greenberg. 1985. Activation of rat and human alveolar macro-phage intracellular microbicidal activity by a preformed LGLcytokine. J. Immunol. 135:1194-1200.

7. Griffin, J. D., T. Hercend, R. Beveridge, and S. F. Schlossman.1983. Characterization of an antigen expressed by human natu-ral killer cells. J. Immunol. 130:2947-2951.

8. Grimm, E. A., K. M. Ramsey, A. Mazumder, D. J. Wilson, J. Y.Djeu, and S. A. Rosenberg. 1983. Lymphokine-activated killercell phenomenon. Precursor phenotype is serologically distinctfrom peripheral T lymphocytes, memory cytotoxic thymus-

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