JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 16:7-16 (1996)Mary Ann Liebert, Inc.

Stimulation of Natural Interferon-al/3-Producing Cells byStaphylococcus aureus

HELENA SVENSSON,1 BRITA CEDERBLAD,1 MATS LINDAHL,2 and GUNNAR ALM1

ABSTRACT

Human peripheral blood mononuclear cells (PBMCs) produced high levels of antiviral activity, as determinedby bioassay, when stimulated by Staphylococcus aureus Cowan I (SAC) and E. coli. Specific immunoassaysdemonstrated the presence ofboth IFN- and and, for SAC, also low levels of IFN-/3. The frequencies ofSAC-induced IFN-a-producing cells (IPCs) were up to 1-2 per 103 PBMCs. These IPCs expressed the HLA-DR andCD4 antigens but not CD3, CD14, or CD19, thus resembling the natural IFN-a-producing cells (NIPC). TheSAC was more efficient as IFN inducer when heat killed than when streptomycin inhibited. The SAC was in¬hibitory to vitally induced IFN- responses, in particular when streptomycin inhibited. Both pronase treatmentand mechanical disruption of SAC cells abolished their capacity to induce IFN- production. Staphylococcalstrains lacking or expressing low levels of protein A (SpA) showed a decreased ability to induce IFN- produc¬tion. However, purified SpA did not itself induce IFN- . Possibly, SpA together with other bacterial surface pro¬teins is important for the capacity of SAC to induce IFN- production in NIPC.

INTRODUCTION

Bacteria can induce the production of interferons (IFNs) invarious cell types in vitro and in vivo, and such IFN might

also be important in resistance to bacterial infections in vivo(reviewed in Ref. 1). In vitro, both Escherichia co/i'2' andStaphylococcus aureus strain Cowan I (SAC) have been shownto stimulate the production of IFNs by blood mononuclear cells(PBMCs). Attempts to characterize the IFN response to SAC im¬plicated that mainly IFN- was produced.'3·4' This IFN- was

acid labile,'4-6' however, and a major part of the IFN activity was

neutralized by antibodies to both IFN- and .'5·6' These resultsmay well be because all previous studies employed bioassays todetermine IFN concentrations at which IFN- , IFN- , and othercytokines, such as tumor necrosis factor (TNF- ), can syner-gize.'7-9' In the present study we therefore used immunoassays to

identify and quantitate more precisely the types of IFN inducedby gram-positive (SAC) and gram-negative (E. coli) bacteria.

Previous work indicated that bacteria induce IFN- produc¬tion in null lymphocytes,'4' not identical to natural killer (NK)cells.'6' The predominant IFN-a-producing cells (IPCs) amongleukocytes exposed to many viruses are infrequent but highlyproductive cells, termed natural IFN-o//3-producing cells(NIPCs; reviewed in Ref. 10. Their phenotype resembles that ofthe null lymphocytes just mentioned, lacking markers of and

cells, monocytes, and NK cells but expressing certain othermarkers, including CD4 and major histocompatibility complex(MHC) class II molecules. We therefore examined whether theIPCs stimulated by SAC have characteristics of NIPCs, with re¬

gard to both frequency and phenotype.Regarding their ability to induce IFN- production, only heat-

killed and formalinized bacteria have previously been studied.We therefore examined the IFN- response to streptomycin-in¬hibited bacteria. For further characterization of the IFN- re¬

sponse, SAC were chosen and tested both as heat killed and as

alive but growth inhibited. In addition, crude fractionations ofSAC were examined. Because staphylococcal protein A (SpA)in one study'11' but not in another*3' was reported to be an IFN- inducer, we reexamined this issue using pure SpA and staphylo¬coccal strains with varying SpA expression.

MATERIALS AND METHODS

Preparation ofPBMCs

Human PBMCs were prepared by Ficoll-Hypaque(Pharmacia, Uppsala, Sweden) density gradient centrifugationof buffy coats or heparinized blood from normal blood donors.The cells were washed three times in phosphate-buffered saline

Department of Veterinary Microbiology, Divisions of 'Immunology and 2Bacteriology, Swedish University of Agricultural Sciences, BiomedicaiCenter, Uppsala, Sweden.

SVENSSON ET AL.

(PBS) and resuspended in RPMI 1640 medium (FlowLaboratories, Irvine, UK) supplemented with streptomycin (100/xg/ml), L-glutamine (2 mM), 20 mM HEPES, and 10% heat-in¬activated fetal calf serum (GIBCO, Paisley, UK).

Depletion ofCD14-positive cells was performed by immuno-magnetic separation according to the manufacturer's recommen¬

dation (Dynal AS, Oslo, Norway). In brief, PBMCs (3 X

106/ml) were incubated with 20 µß/ of a murine anti-CD 14monoclonal antibody (MAb) designated IV:414, produced in our

laboratory. This MAb was shown to specific for CD14 by FACSanalysis and by western blot (not shown). After washing,PBMCs were incubated on ice with Dynabeads M-450 (DynalAS) coated with goat antimouse IgG, and the CD14-positivecells were removed by magnet. This procedure reduced the pro¬portion of CD14-positive cells from 20% to less than 2% for alldonors, according to FACS analysis.

Bacterial strains and cultivationThe staphylococcal strains were SAC, SA 113 (strain 8325-4

lysogenized with phage 83A), prA 3 (producing high amounts ofSpA), and prA 26 (producing low amounts of SpA). The last twoare NTG mutants of SA 113.'12' Protein A-defective 5. aureus

mutants 8325-4 Aspa::Etbr (DU 5273)'13' and NewmanAspa::Tcr (DU5873), and their wild-type parent strains, were

kind gifts from Professor Timothy Foster, Moyne Institute(Dublin, Ireland). The E. coli strain used was NCTC 8623.

Overnight cultures of bacteria on blood agar were used to in¬oculate tryptic soy broth (TSB; Difco Laboratories, Detroit, MI)and grown at 37°C overnight in a rotary shaker. The culture was

used as a 10% inoculum for culture in prewarmed TSB for either2.5 h (logarithmic phase) or 16 h (stationary phase). Bacteriawere harvested by centrifugation at 4000 X g for 15 minutes, re¬

suspended to the original volume, and washed three times in 0.15M NaCl. The resulting pellets were resuspended in 0.15 M NaClwith or without 1 mg streptomycin sulfate (Sigma, St. Louis,MO) per ml, giving a 10 times concentrated bacterial suspen¬sion. Bacterial suspensions without streptomycin (about 5 ml)were heat treated for 5 minutes in a water bath at 80°C. The con¬

centration of bacteria was estimated using a Biircher chamber.Bacteria were diluted in RPMI 1640, supplemented as describedabove to the desired concentrations for further use as IFN induc¬ers. The growth of bacteria was inhibited by the use of strepto¬mycin to prevent overgrowth and production of acid fermenta¬tion products, which could severely and unpredictably affect thefunctions of the PBMCs.

Endotoxin-free, pure protein A (Pharmacia) was used at con¬

centrations of 0.1 ng to 100 µ-g per ml.

Pronase treatment and mechanical disruption of SACSAC from 2.5 h TSB cultures were washed as before and re¬

suspended to a concentration of 5 X 10'" per ml in 0.15 M NaCl.Two of four aliquots (2 ml each) were heat treated as described.Bacteria were then pelleted by centrifugation and suspended in 2ml of 50 mM Tris and HC1, pH 8.0, containing 10 mM CaCl2.Bacteria not receiving heat treatment were resuspended in Trisbuffer containing 1 mg streptomycin sulfate per ml. Pronase (2units/ml; Sigma) was added to two aliquots, one of which was

previously heat treated, and bacterial suspensions were incu¬bated for 40 minutes at 37°C. Bacterial cells were washed three

times in 20 ml cold 0.15 M NaCl as before and resuspended in 2ml of 0.15 M NaCl. Pronase-treated as well as untreated controlbacteria were diluted to the desired concentrations in supple¬mented RPMI 1640.

For the mechanical disruption, SAC from logarithmic growthphase in TSB were harvested and washed as before, and finally a

suspension in 0.15 M NaCl containing 5 X 1010 bacteria per mlwas transferred to one of the compartments of an X-press (Biox,Gothenburg, Sweden), prechilled to

—

35°C. After 2 h the frozenbacterial suspension was disintegrated by nine passages throughthe X-press at 200 MPa. The bacterial paste was thawed and a 2ml suspension was diluted in supplemented RPMI; the remain¬der (8 ml) was centrifuged at 4000 X g and 4°C for 15 minutes toestimate the amount of unbroken cells. The pellet (less than 5%of total amount of bacteria) was discarded and the supernatant,containing cell wall fragments, was centrifuged at 15,000 X gand 4°C for 30 minutes. The pelleted cell wall fragments were

washed three times in 40 ml of 0.15 M NaCl and resuspended in8 ml of 0.15 M NaCl. The yield of resulting cell wall fragmentswas considered to be 95% of the initial amount of cell walls ofthe starting bacterial suspension. Mechanical disruption of heat-killed SAC was not possible using the X-press method.

Virus

Herpes simplex virus (HSV) was adsorbed to human WISHcells (HSV-WISH) and fixed by 0.25% glutaraldehyde, as previ¬ously described."4' Sendai virus (SV) was propagated in eggsand used as chorioallantoic fluid at a dilution of 103 times in thePBMC cultures. Both viruses were used at slightly suboptimalconcentrations with regard to their ability to induce IFN- , to fa¬cilitate any reduction in the IFN response caused by the additionof SAC.

IFN induction

Production of IFN was induced in the PBMCs in 24-well (ex¬periments shown in Tables 1-3) or 96-well plates (Nunclon;Nunc, Roskilde, Denmark) in duplicate or triplicate cultures, re¬

spectively. Volumes were 1 or 0.2 ml, and the final concentrationof PBMCs was 106 per ml. The various IFN inducers were in¬cluded in these cultures, as described. When HSV-WISH was

the inducer, the PBMCs, together with bacteria when indicated,were added directly to the plates containing the WISH cellmonolayers. Culture supernatants were normally collected after24 h of culture at 37°C and .7% C02 in air. No IFN was ever de¬tected in control cultures without IFN inducer(s).

IFN bioassaySupernatants from 24-well cultures were assayed for biologic

IFN activity using human amnion WISH cells as indicator cellsand vesicular stomatitis virus for challenge.'15' Our own internallaboratory standard for IFN- was calibrated against theNational Institutes of Health reference leukocyte IFN- GA-23-902-530. In the neutralization tests, samples were preincubatedfor 1 h at 37°C with 2 103 neutralizing units (NU) per ml of a

sheep antiserum to IFN- , produced in'our laboratory, with 50NU per ml of horse antibodies to IFN-/3 (Boehringer MannheimGmbH; Mannheim, Germany) or with 100 NU per ml of rabbitantibodies to IFN- (Interferon Sciences, New Brunswick, NJ).

STAPHYLOCOCCI AS lFN-cdß INDUCERS

ImmunoassaysThe concentration of IFN- was determined by a dissociation-

enhanced lanthanide fluoroimmunoassay (DELFIA), essentiallyas described previously.'16' The IgG fraction of a bovine anti-serum to human IFN- (AS 94; Wellcome Research Foundation,Beckenham, UK) was treated with 50 mM HC1 at a ratio of 1:6(vol/vol) for 5 minutes at room temperature before dilution. TheAS 94 was then diluted to 5 pg per ml, added to microtiter plates(Maxisorp; Nunc), and incubated overnight. The plates were

washed and the remaining binding sites blocked by incubationwith postcoating buffer.

All samples and standards (see earlier) were incubated in thewells at final concentrations of 50% medium and 5% normalbovine serum (to reduce unspecific binding of SpA) in dilutionbuffer. Following incubation and washing, a mixture of opti¬mized concentrations of europium-labeled MAbs LT 27:293 andLT 27:295'17' in dilution buffer were added. The two MAbs were

labeled by europium lanthanide chelate according to the manu¬

facturer's recommendations (Wallac Oy, Turku, Finland). Thefluorescence was then measured by a time-resolved fluorometer(Wallac) and data analyzed using the Multicalc program(Wallac). The 293 MAb binds to all IFN- subtypes except IFN-a2, and the 295 MAb binds IFN-a2 and several other subtypes.When combined the two MAbs bind all IFN- subtypes, but not

IFN-/3 or , present in the IFN produced by SV-stimulatedPBMCs (unpublished results). It is therefore likely that the ma¬

jority of IFN- subtypes are detected by the described im¬munoassay.

The concentration of IFN-/3 in culture supernatants was deter¬mined by enzyme-linked immunosorbent assay (ELISA).'17'Interferon- was determined by DELFIA essentially as de¬scribed above. Plates were coated with 5 µ% per ml of the MAb6G6.E7 to IFN- , which had been purified by protein G andcation-exchange chromatography. Another MAb, 8B8.B5, was

similarly purified and labeled with europium lanthanide chelate.Purified natural IFN- was used as standard. All reagents to IFN- were kindly provided by Dr. H.-L. Kauppinen (Finnish RedCross Blood Transfusion Service, Helsinki, Finland).

ELIspot assayThe frequencies of IFN-a-producing cells were determined

by the ELIspot assay, as previously described."8' In short,PBMCs were stimulated in 96-well plates (Nunclon) for 8 h bythe indicated IFN inducer, suspended, and transferred in a well-to-well fashion to AS 94-coated nitrocellulose-bottomed 96-well plates (Multiscreen-HA; Millipore Co., Bedford, MA).Following overnight incubation at 37°C, plates were washed, in¬cubated with two peroxidase-conjugated MAbs to IFN- (LT27:293 and LT 27:297, also detecting more than 90% of IFN- subtypes when combined), and washed, and finally the substratediaminobenzidine was added.

In situ hybridizationFor the detection of cells containing mRNA for IFN- , PBMCs

were stimulated by the various inducers for 6 h in 24-well cultures,suspended, and fixed overnight in paraformaldehyde as previ¬ously described."9' In situ hybridization was then performed witha 35S-labeled cRNA probe to human IFN-a2'20' as described.'21'

ImmunohistochemistryThe PBMCs were stimulated with IFN- inducers for 6 h, sus¬

pended, and washed once in PBS before fixation in 1%paraformaldehyde in PBS at 4°C overnight. The cells were

washed twice in PBS with 0.1% human serum albumin (HSA;Kabi-Pharmacia, Stockholm, Sweden), suspended in PBS-HSA at5 106 per ml, and 25 µ\ added to each well (diameter 8 mm) ofglass slides with hydrophobic coating. The slides were dried for 1h at 37°C and stored at

—

80°C. Before staining, cells were rehy-drated in PBS for 5 minutes and endogenous peroxidase blockedby incubation in 0.3% H202 in PBS twice for 15 minutes at RT.Slides were then rinsed in PBS with 0.1% Tween for 5 minutes andunspecific binding sites blocked by incubation in PBS-Tween sup¬plemented with 20% bovine serum and 20% goat serum (DAKO;Denmark) for 20 minutes in a humid chamber. After rinsing inPBS-Tween, cells were incubated for 45 minutes with the indi¬cated MAbs at 10-20 µ% per ml in PBS-Tween supplemented with10% pooled heat-inactivated human AB serum. Slides were rinsedagain, simultaneously incubated for 45 minutes with peroxidase-labeled goat antibodies to mouse Ig (Jackson Immuno Research,Avondale, PA), and FTTC-labeled AS 94 (50 µg per ml) diluted inPBS-Tween-human serum. After rinsing, the substrate di¬aminobenzidine (0.5 mg per ml) and 0.1% H202 in PBS were

added and the reaction terminated after 4—8 minutes by rinsing inPBS. Slides were mounted in Fluoromount G (SouthernBiotechnology Associates, Birmingham, AL) and stored in thedark at 4°C. For each MAb, at least 200 PBMCs were examinedand the percentage of stained cells calculated.

RESULTS

Characterization of the IFNs induced

The PBMC were stimulated in vitro by streptomycin-inhib¬ited SAC or E. coli. Culture media collected after 24 h were ana¬

lyzed for IFN- , ß, and by the use of bioassays, combined withspecific neutralizing antibodies, and by specific immunoassays.

Both SAC and E. coli induced the production of signifi¬cant levels of antiviral activity in the PBMCs (Table 1).Neutralization with antibodies indicated that most of the antivi¬ral activity was caused by IFN- and , with little or no contribu¬tion from IFN-/3. Combination of antibodies to all three types ofIFN completely neutralized the antiviral effects, indicating thatclassic IFNs indeed were responsible for the bioactivity.

Immunoassays demonstrated that actual levels of IFN- and were considerably lower than predicted from the bioassay data(Table 1). The IFN- responses to bacteria were in general muchweaker than those to viral inducers, such as HSV-WISH (3000and 1700 U per ml in donors A and B, respectively). No signifi¬cant levels of IFN-/3 (<5 U per ml) were detected, but in otherexperiments (see Fig. 5) in which SAC induced high levels ofIFN- (mean of 750 U per ml), approximately 30 U IFN-/3per ml was detected.

Kinetics and variability of the IFN- responsesinduced by SAC

The kinetics and variability of the IFN- responses of PBMCfrom eight healthy donors were studied over a 48 h culture pe-

10 SVENSSON ET AL.

Table 1. Characterization of the Antiviral Activity Produced by PBMCs When Stimulated by SAC or E. Coli*

IFN concentration (U/ml) measured by

Inducer

Bioassay*'Control a ß a + ß a + ß+

ImmunoassayIFN-a IFN-

SAC

E. coli

A A

500e90

500400

20050

15090

50080

500350

35045

12560

10030

180150

<10<10<10<10

75<36030

752

1010

aThe PBMCs of two donors (A and B) were cocultured for 24 h with 5 106 streptomycin-inhibited bacteria per ml. The levels ofIFN were then determined by bioassay and by specific immunoassays. No IFN-jS was detected in these experiments (<5 U/ml), andno IFNs were detected in control cultures without bacteria.

bAntiviral activity was determined by bioassay in the absence (control) or presence of neutralizing antisera to IFN- (a), IFN-/3(ß), IFN- ( ), or indicated combinations thereof. See Materials and Methods for further details.

"Figures are mean of duplicate cultures.

riod. Here and in later work, IFN concentrations were deter¬mined by immunoassay. Both streptomycin-inhibited and heat-killed SAC were used for induction of IFN- , and for compari¬son the viral NIPC inducer HSV-WISH was also included.

For all donors, streptomycin-inhibited bacteria were less effi¬cient inducers of IFN- production than heat-killed bacteria(Fig. 1 ). The IFN- levels obtained with the latter were 10-20%of those obtained with HSV. However, the kinetics of the IFN- responses were similar; that is, most IFN- were producedwithin 20 h after addition of the inducer. In contrast, a continuedIFN- production beyond this time was observed for some

donors using streptomycin-inhibited bacteria as inducer. For allIFN inducers, considerable differences between donors were

seen with regard to the magnitude of the IFN- response. For in¬stance, at 20 h of culture the IFN- production in response to

streptomycin-inhibited SAC ranged between 40 and 620 U/ml(median 140 U/ml), to heat-killed SAC, 80-1730 U/ml (median390 U/ml), and to HSV, 920-7500 U/ml (median 3000 U/ml).No correlation for the individual donors was observed betweenmagnitudes of HSV- and SAC-induced IFN- responses.

Frequencies and productivity of SAC-inducedIFN-a-producing cells

Frequencies of SAC-induced IPCs were estimated by RNA-RNA in situ hybridization, by immunofluorescence of intracel-lular IFN- (IF), and finally by ELIspot demonstrating actualIFN-a-secreting cells. These assays revealed that the IPCs were

infrequent among SAC-stimulated PBMCs (Table 2). The num¬

ber of IPCs induced by heat-killed SAC was estimated to be15-20 IPC per 104 PBMCs by in situ hybridization and ELIspotassay, and lower estimates were obtained by the immunofluores¬cence technique. Stimulation of PBMCs by streptomycin-inhib¬ited SAC generally resulted in lower IPC frequencies than didheat-killed SAC. Using IPC frequency data and immunoassayestimates of total IFN- content in culture media, the amount ofIFN- produced by each IPC was calculated to be of the order of1 U. Furthermore, for both streptomycin-inhibited and heat-killed SAC, the corresponding IPCs showed very strong hy¬bridization signals, intense immunohistochemical staining, andlarge ELIspots (results not shown).

Antigenic phenotype of SAC-induced IPCs

The phenotype of SAC-induced IPCs was studied using im-munoperoxidase staining for antigens characteristic of cells(CD3), cells (CD19), and monocytes (CD14), as well as CD4and HLA-DR. The last two are expressed on NIPC.'10' The IPCswere identified by immunofluorescence using FITC-labeled an¬

tibodies to intracellular IFN- .This double-labeling technique revealed that a clear majority

(>80%) of the SAC-induced IPCs expressed CD4 and HLA-DRbut not CD3, CD19, or CD14 (Table 3). However, both strepto-

10000 -,

1

Uh

1000

100-

10

I10

20

— -30 40

- 50

Culture time (h)FIG. 1. Kinetics of the IFN- response in PBMCs induced bystreptomycin-inhibited SAC (squares), heat-killed SAC (dia¬monds), or HSV-WISH cells (circles). The PBMCs of eightdonors were cocultured with the respective IFN inducer for theindicated periods of time. The levels of IFN- were determinedby DELFIA and the median production of the eight donors ateach point of time calculated. See Materials and Methods for fur¬ther details.

STAPHYLOCOCCI AS WN-alß INDUCERS 11

Table 2. Frequencies of IPCs and Amount of IFN- Produced per IPC in Cultures of PBMCs Stimulated byStreptomycin-Inhibited or Heat-Kjlled SACa

IPC frequencies0 determined by Amount of IFN-ac

Inducer ISH IF ELIspot U/ml U/IPC

Str-SACHk-SAC

2.619.7

0.5d3.9

1.66.9

0.40.5

2.614.6

0.81.6

300 ± 501320 ± 160

1.20.9

0.20.2

aThe PBMCs were cocultured with either streptomycin-inhibited (Str) or heat-killed (Hk) SAC at 5 X 107 bacteria per ml in dupli¬cate cultures of 24-well plates.

bFrequencies of IPCs were determined by in situ RNA-RNA hybridization (ISH), immunofluorescence (IF), or the ELIspot assayin PBMCs following 6 h (ISH, IF) or 8 h (ELIspot) of culture. Frequencies are expressed as number of IPCs per 104 PBMCs.

cLevels of IFN- were determined by DELFIA in 24 h culture supernatants. Mean amount of IFN- produced per IPC was calcu¬lated from the total amount of IFN- produced after 24 h of culture and parallel ELIspot estimations of IPC frequencies.

dResults are mean ± standard deviations (SD) of three donors.

mycin-inhibited and heat-killed SAC clearly downregulatedCD 14 on PBMCs and could have the same effect on any CD 14on IPCs. Therefore, PBMCs were depleted of cells expressingCD 14 by immunomagnetic separation before IFN induction bySAC. Such CD14-depleted cells, stimulated by streptomycin-in¬hibited SAC, showed at least a twofold increase in IFN- pro¬duction, compared with unseparated PBMCs (Fig. 2). In con¬

trast, their IFN- response to heat-killed SAC was unaltered or

showed a smaller increase. Therefore, the IPCs appeared CD 14negative and, with regard to expression of all investigated mark¬ers, resembled NIPC.

Effects of SAC on virally induced IFN-a responsesHeat treatment of SAC increased their capacity to induce IFN-

a (see Fig. 1), which indicates a potential capacity of strepto¬mycin-inhibited SAC to counteract the IFN- response. Wetherefore examined the effects of heat-killed and streptomycin-inhibited SAC on the IFN- responses induced by HSV inNIPC*22·23' and by Sendai virus predominantly in mono¬

cytes.'23·24'Streptomycin-inhibited SAC caused up to a 50% reduction in

the HSV-induced IFN- response (Fig. 3A). This was seen evenat bacteria/leukocyte ratios between 0.5-5 and 1. At the same

concentration, heat-killed SAC had no inhibitory effect, but atthe highest SAC concentration tested, heat-killed and strepto¬mycin-inhibited bacteria were equally inhibitory. In contrast,both streptomycin-inhibited and heat-killed SAC equally andstrongly inhibited the IFN- response induced by Sendai virus(Fig. 3B).

IFN- inducing capacity of SAC at differentgrowth phases and after protease treatment or

mechanical disruptionSAC was harvested in the logarithmic growth phase (2.5 h)

and in the stationary phase (16 h). Such bacteria, streptomycininhibited or heat killed, were tested as IFN- inducers. In addi¬tion, the effects of pronase treatment on their IFN- inducing ca¬

pacity was determined.Streptomycin-inhibited SAC obtained during exponential

growth were much more potent IFN- inducers compared withSAC in the stationary phase, especially at high SAC concentra¬tions (Fig. 4). In contrast, when the two types of SAC were heatkilled, they induced both high and similar IFN- responses.Also, the maximal IFN- response when using heat-killed SACwas obtained at a lower SAC concentration than when usingstreptomycin-inhibited SAC. These results indicate that the

Table 3. Antigenic Phenotype of IPCs and PBMCs Induced by Streptomycin-Inhibited or Heat-Killed SACa

% Ag-positive cellsb in

Cell surface Ag

IPCs Total PBMCs

Str-SAC Hk-SAC Medium Str-SAC Hk-SAC

CD3CD4CD14CD19HLA-DR

0 (32)c83 ± 10 (32)

0(36)0(37)

82 ± 9 (30)

0(74)79 ± 4 (96)

0(79)0 (124)

80 ± 18 (102)

57 ±341 ±526 ±38± 1

24 ±5

73 ± 150 ± 13

3 ± 19± 3

13 ±0

72 ±354 ±96±27±2

13 ±3

"PBMCs were cultured in duplicates of 24-well plates for 6 h in the absence (medium) or presence of 5 X 107/ml streptomycin-in¬hibited (Str) or heat-killed (Hk) SAC and then processed for subsequent double labeling of cell surface antigen and intracellular IFN- by immunohistochemistry (see Materials and Methods).

Percentage of Ag-positive IPCs and PBMCs, respectively.Tigures are mean ± SD of three donors. The total number of evaluated IPCs from the three donors within parentheses.

12 SVENSSON ET AL.

600 Str-SAC and:

O Unsep PBMCs

G CD14-depleted PBMCs

Hk-SAC and:

Unsep PBMCs

CD14-depleted PBMCs

FIG. 2. Effects of depletion of CD14-positive cells on the in¬duction of IFN- by streptomycin-blocked (Str) or heat-killed(Hk) SAC. The PBMCs of three donors ( -C) were depleted ornot of CD14-positive cells and then cocultured with 5 X 106bacteria/ml for 24 h. The IFN- levels were determined byDELFIA. Results are mean ± SD of triplicate cultures for eachdonor.

mode of preparation of SAC is a significant cause of variation intheir IFN-a-inducing capacity. As further shown in Fig. 4,pronase treatment destroyed most of the IFN-a-inducing capac¬ity of SAC, suggesting that SAC surface proteins are importantfor the IFN-a-inducing capacity of SAC.

In attempts to define subcellular interferogenic components ofSAC, bacteria were mechanically disrupted by an X-press. Thispreparation had a dramatically reduced IFN-a-inducing capacitycompared with intact streptomycin-treated SAC (Fig. 5). Also,washed cell walls, prepared from the mechanically disruptedSAC, lacked IFN-a-inducing capacity (not shown). Assays ofIFN-/3 were also performed in these experiments, with similarresults as with IFN- , although IFN-/3 levels in general were

much lower (Fig. 5).

Role of SpA in the induction of the IFN- responseby SAC

Protein A (SpA) of SAC has been shown to induce IFN- pro¬duction in human leukocytes."" We failed to confirm these re¬

sults using highly purified SpA (Table 4), which could be a resultof differences in the SpA preparations. Therefore, we investi¬gated staphylococcal strains with varying expression of SpA fordifferences in IFN-a-inducing ability. When using heat-killedbacteria as IFN- inducers, there was a clear indication thatstrains with low SpA expression (prA 26) and the two SpA nega¬tive mutants of strains Newman and 8325-4 were considerablyless potent IFN- inducers than the relevant SpA-positive strains(Table 4). These results suggest that SpA may in some way con¬

tribute to the IFN-a-inducing ability of SAC. However, all strep¬tomycin-inhibited strains of S. aureus, except Cowan I, were

poor inducers of IFN- , and no relation between expression ofSpA and IFN- responses was seen (results not shown).

DISCUSSION

Studies of IFN production by leukocytes have so far verymuch focused on stimulation by viruses; less is known about

bacteria as stimulators. In this study we demonstrate, by specificimmunoassays, that both gram-positive (here, SAC) and nega¬tive (E. coli) bacteria stimulate the production of IFN- and inleukocytes. We further examined the IFN- response to SACwith regard to the identity of the actual IFN-a-producing cell andfactors influencing the IFN-inducing ability of SAC.

By bioassay we confirmed previous results'2^1' demonstratingthe appearance of relatively high levels of antiviral activity incultures of PBMCs stimulated by either SAC or£. coli. This an¬

tiviral activity was completely dependent on the presence oflFN-a/ß and , because it was neutralized by specific anti-IFNantibodies. However, the actual concentration of IFNs deter¬mined by immunoassays were usually much lower, 10-fold or

more, than indicated by bioassay data. These differences couldnot be explained by partial detection in the immunoassays, not

150-,

1002Pi

J-, 50->

Str-SAC

Hk-SAC

0.1 10

106 SAC per ml

150-

100-

100

2ti

TJ 5°·>

0.1 10 100

6 SAC per ml

FIG. 3. Effect of streptomycin-inhibited or heat-killed SACon the IFN- responses of PBMCs induced by HSV-WISH (A)or Sendai virus (B). The PBMCs were cocultured for 24 h withHSV-WISH or Sendai virus in combination with the indicatedconcentrations of streptomycin-inhibited (filled squares) orheat-killed (open circles) SAC. The IFN- levels were deter¬mined by DELFIA and are expressed as percentage of the IFN- levels obtained with the viral inducers alone (2600 U/ml forHSV-WISH and 4300 U/ml for SV). Results are mean ± SD ofseven (A) and four (B) donors, respectively.

STAPHYLOCOCCI AS WN-a/ß INDUCERS 13

Str-SAC:

Log-phaseStat-phaseLog-phase,pronase treated

Hk-SAC:

Log-phaseStat-phaseLog-phase,pronase-treated

Table 4. Influence of the Expression of Protein A by

Various Strains of S. aureus on the Induction of IFN- Production in PBMCs"

10" SAC per ml

FIG. 4. Capacity of SAC harvested at logarithmic (Log) or

stationary (Stat) growth phase, treated or not with pronase, to in¬duce production of IFN- . Both streptomycin-blocked (Str) andheat-killed (Hk) SAC were tested. The PBMCs were coculturedfor 24 h with SAC as indicated, and IFN- levels were deter¬mined by DELFIA. Results are mean ± SD of five donors.

even for IFN- , because the antibodies used in the DELFIA de¬tects essentially all the IFN- subtypes (see Materials andMethods). Instead, the discrepancies between bioassay and im¬munoassay results are most likely caused by a potentiation of thebiologic activities of the IFNs. Thus, synergy between IFN-a//3and IFN- have been reported,'7·25' and the IFNs may also syner-gize with other cytokines, such as TNF-a,'8·9·26' Such synergiesmay also explain observations in previous studies using SAC'5·6'and/or E. coli,w in which atypical acid-labile IFN- and IFNwith common antigenic properties of IFN- and were demon¬strated by bioassays and neutralizing anti-IFN antibodies.Compared with immunoassays, bioassays therefore providepoor estimates of the true concentrations of IFN induced by bac¬teria. On the other hand, the enhanced biologic activities of IFNseen with bacterial inducers may be biologically relevant in an-

1250

1000·

750DÖ

I

[i

IO6 SAC per ml

FIG. 5. Effect of disruption by X-press of streptomycin-inhib¬ited SAC on their ability to induce IFN- and ß production. ThePBMCs were cocultured for 24 h with indicated concentrationsof streptomycin-inhibited SAC, either intact (open symbols) or

mechanically disrupted by X-press (filled symbols). Levels ofIFN- (squares) were determined by DELFIA and of IFN-/3 (cir¬cles) by ELISA. Results are mean ± SD of three donors.

Amount of IFN-a (U/mlfS. aureus strain C

Experiment 1Cowan I 300 420 NDCSA 113 wt 100 70 NDSA 113 prA 3 390 200 NDSA 113 prA 26 30 40 ND

Experiment 2Cowan I 610 440 170Newman wt 60 80 6Newman SpA" 20 10 <68325-4 wt 120 220 608325-4 SpA" 20 10 <6Protein A (100 /xg/ml) <6 <6 <6

"The PBMCs of three donors ( -C) were cocultured for 24 hwith the various strains of SAC at 5 X 106 bacteria per ml or withprotein A (0.1 ng/ml to 100 /¿g/ml tested with identical results).All bacteria were heat killed.

bLevels of IFN- were determined by DELFIA and are ex¬

pressed as mean of triplicate cultures of each donor.cNot done.

tibacterial immune responses, given the many immunomodula-tory actions of the IFNs.

The actual IFN-a-producing cell among PBMCs stimulatedby SAC appeared similar to the natural IFN-a/ß-producing cells.The NIPC was previously determined to be the main IFN- pro¬ducer stimulated by HSV and several other viruses (reviewed inRef. 10).(27) Thus, the frequency of IPCs induced by heat-killedSAC was low (1-2 per 103 PBMCs) in donors having relativelyhigh IFN- responses, as determined by in situ hybridization or

ELIspot (Table 2). Both these methods detect essentially allNIPCs."4' These data were confirmed using the apparently lesssensitive immunocytochemical staining for intracellular IFN- .Furthermore, the corresponding IPCs showed very strong hy¬bridization signals, intense immunohistochemical staining, andlarge ELIspots, that is, results comparable to those obtained withHSV-WISH as the IFN- inducer. Each SAC-induced IPC ap¬peared to produce of the order of 1 U IFN- , an estimate similarto that of HSV-stimulated NIPC."4·20·28' Also the antigenic phe¬notype of the SAC-stimulated IPC resembled that previously es¬

tablished for NIPC (for review see Ref. 10). Thus, the SAC-in¬duced IPCs did not express the CD3 of cells, CD 19 of cells,or CD14 of monocytes, but the majority expressed CD4 andMHC class II antigens. Depletion ofCD14-positive cells demon¬strated, despite a great variability among donors, that these cellsare not essential for the production of IFN- in response to stim¬ulation by SAC. However, we cannot rule out that SAC may in¬duce IFN- in CD14-negative monocytes,'29' because mono¬

cytes have been reported to produce IFN- in response to Sendaivirus'23·24' and Corynebacterium parvum.m

The magnitude of IFN- responses to SAC varied consider¬ably between individual PBMC donors, with up to 10-fold dif-

14 SVENSSON ET AL.

ferences in IFN- levels as determined by immunoassay.Differences were also seen between individuals with regard toIFN- responses to the viral inducer HSV. The reason for thisvariation could in principle be caused by different numbers ofpotential NIPC or differences in essential cofactor produc¬tion."7·3" A large individual variation in NIPC frequencies forviral inducers has also been demonstrated.'14·28·32' When compar¬ing the magnitude of SAC- and HSV-induced IFN- responsesfor each individual donor, no correlation was found. The lack ofcorrelation could be a result of different mechanisms of trigger¬ing of NIPC by these IFN- inducers.

Streptomycin-inhibited SAC caused a significant decrease inthe HSV-induced IFN- response and were also less potent IFN- inducers than heat-killed bacteria. In addition, supernatants re¬

trieved from SAC upon heat treatment reduced the IFN- re¬

sponses to both virus and bacteria (results not shown). Theseresults suggest the existence of cell wall components that coun¬

teract the IFN- response and are removed from SAC upon heattreatment. Heating has been shown to induce loss of ester-boundalanine residues of cell wall teichoic acids.<33) Surface proteins ofS. aureus may be bound to teichoic acids,'34' but the nature of thislinkage is still unclear. However, alanyl residues of teichoicacids may be involved in the binding of proteins to the bacterialcell wall, either by covalent bonds or by electrostatic interaction.Ester-bound alanine serves, in the case of non-covalently boundcell wall proteins, as the sole positive charge of teichoic acid andmay therefore be crucial to binding of negatively charged sur¬

face proteins.In contrast, the heat-sensitive components are not essential for

the SAC-mediated inhibition of the SV-induced IFN- responseof monocytes, because both streptomycin-inhibited and heat-killed SAC were equally inhibitory. In fact, previous observa¬tions indicate that the IFN- response of monocytes is inhibitedby phagocytosis of particles.'23'

Activation of monocytes-macrophages by SAC may partlyexplain both the low IFN- responses induced by SAC and theability to suppress HSV-induced IFN- responses. Supportingthis view, the IFN- response induced by streptomycin-inhibitedSAC was approximately doubled when PBMC were depleted ofthe CD14-positive monocytes. These results are also in line witha previous observation that adherent cells suppressed SAC-in¬duced IFN- response in PBMC.'6' However, the SAC may alsodirectly interact with NIPC via various surface structures, some

lectin-like, and cause suppression. Thus, several plant lectins'35'and antibodies to cell membrane structures"8·36' efficiently sup¬press IFN- responses ofNIPC.

Streptomycin-inhibited SAC was a much more potent IFN- inducer when harvested during exponential growth comparedwith SAC at stationary phase. In contrast, equally good IFN- responses were obtained with heat-killed SAC regardless ofgrowth phase. These results suggest that changes in bacterialproperties during their growth, such as expression of surfacestructures,'37' influence their ability to stimulate or inhibit IFN- responses in NIPC. In particular, the ability of growing bacteriato inhibit potentially immunostimulatory WN-a/ß responsesmay be of pathogenetic significance (see later).

The complete abrogation of SAC-induced IFN- response bypronase treatment strongly indicates a proteinaceous nature ofthe interferogenic structures of SAC. Protein A appears to be one

of the interferogenic proteins of S. aureus, because mutants lack¬

ing SpA induced less IFN- than corresponding parent strains.However, pure SpA did not induce IFN- . Furthermore, SACappeared to lose the IFN-a-inducing ability upon mechanicaldisruption. Apparently, the interferogenic structures must bepresented on a particulate phase, that is, on intact bacteria.

Regarding the mechanism(s) whereby the IFN- response ofNIPC is triggered, it has been suggested that such cells recognizeinterferogenic structures on virus or virus-infected cells.'38-41'Concerning bacterial IFN- inducers, the results of the presentinvestigation suggest that interaction(s) with bacterial surfacestructures, such as SpA, is important. However, other interfero¬genic components of staphylococci must also exist becausestrains lacking SpA, or with low SpA expression, still conveyconsiderable IFN- induction.

The ability of SAC either to stimulate or inhibit production ofIFN-0//3 by NIPC may be biologically relevant for several rea¬

sons. The NIPC resemble the important antigen-presenting den¬dritic cells regarding both antigenic phenotype'42·43' and the abil¬ity to present antigens to cells (Cederblad et al, manuscript inpreparation). Following administration of virus in vivo, IPCs(probably NIPC) are mainly found in lymphoid organs near or inareas of antigen-presenting cells"6·21' (M.-L. Eloranta, manu¬

script in preparation). Further, a possible important immunoreg-ulatory role of IFN- is supported by the ability of IFN- to pro¬mote the development of helper 1 cells and cytotoxic cells,'44·45' as well as to regulate cell migration in lymphoid or¬

gans.'46·47' The influence of IFN-cdß on the development of im¬mune responses to bacteria is poorly understood. However, it isreasonable to believe that the bacterial impact on the IFN-a//3production affects both antigen presentation and development of cell responses and may therefore be of vital importance to thepathogenesis ofbacterial infections.

ACKNOWLEDGMENTS

We thank Kristian Sandberg for performing the ISH and our

collaborators at the laboratory for providing us with their blood.This study was supported by grants from the Swedish MedicalResearch Council.

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Address reprint requests to:Dr. Brita Cederblad

Immunology (V)Biomedicai Center, Box 588

751 23 Uppsala, Sweden

Received 1 June 1995/Accepted 15 August 1995